I also had a couple criteria for myself. The guest had to appear in more than two episodes, and I don't include Jim Beaver just because he's more of a regular to me than a guest star. I may have missed some of your favorites, but I tried to go with what I thought were the most universally beloved, the ones that everyone gets pumped about when they would see his or her name at the top of the hour.
These aren't in any particular order (I included a link to one of my favorite scenes from each guest beneath the photo)...
The erstwhile Ash makes this list based on his ridiculously embraced mullet alone. You can (or could) always rely on him to bring a lighthearted tone to whatever dire situation they may have been in, which was always a breath of fresh air.
While the yellow-eyed demon was a constant background presence for most of season one, he gained a face in season two when Fred Lehne brought him to life. He seemed to have a lot of fun playing the demon, and it made us love to hate him.
A lot of people have basically cemented their belief that Chuck was actually God, but I'm not so sure. I prefer him as the goofy Chuck, the prophet and writer of the Supernatural novels. While it could be argued that bringing in that meta-type story was kind of weird, you can't deny that Chuck was entertaining, and I think we can thank Rob Benedict a lot for that.
Trickster turned angel Gabriel. I'm not sure I really liked the twist that he was actually an angel in hiding. I preferred him when he was just the trickster up to no good. But hey, a prime example of a guest star being so popular that his role was expanded.
Women have a hard time in the Supernatural world. Unless the woman happens to be older and more of a mother-type. And Ellen fits that bill completely. As played by Samantha Ferris, Ellen was hardcore but with a soft heart, and it's always a pleasure to see her, even though she may be technically dead now. Come to think of it, most of these guest stars' characters are dead!
From the first time Colin Ford played young Sam back in season three, it became clear that he was perfectly cast. I know personally I get really excited every time he's going to be on the show. Not only because that means we get to see a flashback moment (most of the time), but because I love his take on the young Sam.
Mary Mary, quite contrary, how does your garden grow? Okay, sorry, I don't know what I'm doing. Mary is not a very contrary character in the Supernatural world, and it's always great to see her come back. I went with Samantha Smith instead of Amy Gumenick because when you think of Mary, you think of Sam. At least, I do. Though she had a secretly badass past to her, the Mary Dean knew as a kid was nothing but his mom, and... ahem. Awww.
By far the guest star I most wish the show could get back. (And can I just complain for a second how epically long it took me to just find that damn clip?! You'd think that would be an easily searchable clip but no!) ANYWAY. John Winchester was a polarizing figure in the boys' lives. He made them the hunters they are today, but that led to a lot of resentment. I really wish JDM could come back for at least some flashback episodes or something, because seeing him and Colin Ford together would be pretty epic, wouldn't it? Or how about him and Samantha Smith? I'm not sure they even really had a scene together in the pilot at all! Sad.