I may not do much greater shading, but there will be additional shadows that aren't in this image. Shadows are going to be done is a separate layer by applying 30% translucent solid black shapes. There's some shadowing built into the tileset (like the rooftop ledge) but for the most part, it'll be done separately. I have not tried this technique yet, so I don't know how well it will work. I'm hoping it will be something that will save considerably on time and extra tiles.

I agree that the roads look odd. If you'd care to elaborate, maybe that would help me figure out why they look odd.

Texture, contrast, and heavy shading are a stylistic choice I'm avoiding. I want to minimize such things as much as possible to get a more cartoony, Earthbound feel.

This is a daytime scene. For nighttime scenes, I'll apply a 20% navy blue overlay over the top of it. This project would feature the town at various levels of zombie infestation, so while there will be tiles for boarded up windows and barricades, I'm starting with the town before a zombie attack so that these other additional features may be built on top of it. I'm hoping for a one to one replacement deal, so that I can replace an open window with a boarded up window, or door with a barricaded door and so on. But that's for later. Nighttime will be accomplished with a 20% navy blue layer applied on top.

The facial expressions on the plant are very emotive. Very good. However, I'm a little concerned about the mouths on the people. I'm not sure that you'll be able to have enough definition there to get the different between a smirk and a smile. With the woman in white, the mouth is too close to the chin area, which has a lot of dark pixels, making it very difficult to make out the expression (if you are going 2x pixels, this probably won't be as much a problem).

The black outline and light shading will make a huge difference when you try to place these characters against a bunch of different backgrounds. What works against a dark busy background doesn't always work against a light solid color, so you really need to show these characters off against several different backgrounds to make sure they work. The definition of the plant's stem could be lost in a busy dark background, for instance, so you might want to use lighter colors there to make it pop out more. Only way to know is try.

Pixel art comics... what will they think of next...? 

What is your point Sqorgar?   

The part where he is supposedly walking at night, has an orange color - the color you would give something during a sunrise or sunset. Then, supposedly after the sun is in the process of rising, it goes to a very dark highlighted purple, which gives it a nighttime feeling. It's like Batman: The Animated Series. They drew on black paper to give the show a dark feeling. But sunrise isn't dark. It's orange first, and then moves lighter and lighter to a whitish color, then light blue to sky blue. On cloudy or winter days, the sky doesn't go blue so much as bluish gray, where the gray gets progressively lighter and bluer. But under no circumstance, does it EVER get darker. It does not go from orange to purple. It goes from purple to orange.

The comic would work much better if he switched the words to match the chronology of color. He goes walking in the twilight, and after sunset, crap happens. But then, the owl still wouldn't have any feet.

I'm really not sure what to do about the broken images.  It works fine in Firefox, is it my school's server or something?

IE doesn't properly recognize PNG files with alpha transparency. Save it again in 8-bit or 24-bit rather than 32-bit.

No, it does turn it a pinkish blue colour though.

And it does this before or after the part where everything is orange?

Ive been doing experiemnts with the outlines/inside lines of the characters, and so far i like the results.  here is an animated gif, and i think you can see which is the newer:

I also have been experimenting with the tiles, and am equally pleased:

I think the difference is like night and day. The soft internal lines with the hard outline really make a huge amount of difference. For the tiles, the shading literally brings out the definition. The one thing you should think about is making the ramp tiles noticably darker than the flat ground tiles, and make ramps one direction noticably darker than the other. The definition of the land is literally defined by the negative space. You aren't creating the three dimensional shape by what's there, but from the shadows themselves. As such, the ramps create sort of a muddled look - especially towards the bottom where you've got two ramps going two different directions on two different planes. It creates a visual confusion - but that may just be because you've got a ramp leading up into a mismatched ramp, so it doesn't flow together as well.  The ramps are not so much an equal part of the landscape, but a transition between layers.

It's like... have you ever seen a map for a roguelike game? Most of them represent it as simple gray squares with something like blue doors. But if you don't fill in the doors and just use a floor tile, the map looks over busy and kind of... spirally, if that makes any sense. If you fill in doors with wall tiles, the map feels too claustrophobic. But explicitly defining the doors as neither wall nor floor, you create this sort of node based map with connectivity. It compartmentalizes each room while still creating a distinct impression of how the floorplan flows. It's really weird to explain though.

And you might consider - I've never tried this out nor know if your engine can handle it, so this is just a brainstorm thing - adding a 1%-2% darkening to each layed (0% on the top layer, 2% on the next down, 4% on the one below that) creating a sort of darkening depth thing. That may look funny doing it dynamically, but I've created some (non-isometric) game art that did it manually - I knew the floor would always be exactly four tiles back, so I had a different set of tiles at a different darkness and scale to give the illusion of depth... plus, it paralax scrolled really nicely. The trick is, and I've gotten in trouble for saying this before, to make it almost invisible to the naked eye but explicit enough that the effect works.

i believe i mentioned earlier that i was working on tall grass, and you can see some of it in the first picture, but it is also a walkable tile that the player can move between, and it will be animated.  here is a still shot of characters standing in the tall grass:

I don't have time to comment on this right now, but I'd recommend making the grass shorter (about waist high) and use a harder outline where it overlaps the characters - not black, but a harder dark green, maybe.