- Homestar Runner:
- Strong Bad started out as a villain who was always doomed to lose to Homestar. Now he's debatably the most popular character, and definitely one of the most recognisable.
- To a lesser extent, there's Homsar. He first appeared as a one-shot gag to make fun of a typo, and over time, has been promoted up the ranks to become part of the main cast. HR's creators have joked about avoiding giving Homsar his own cartoon since they would never need to use him again.
- Same goes for Senor Cardgage.
- Trogdor and Stinkoman (Strong Bad's blue-haired anime counterpart) are also very popular with the fans. They even appear together in the fifth episode of SBCG4AP!
- Also Eh! Steve from the Show Within a Show Sweet Cuppin' Cakes. Strong Bad insists that the main character is Sherlock, some kind of "mix between a cow and a helicopter" who is constantly trying (and failing) to get a worm out of the ground, and that Eh! Steve is a background character who delivers his Catchphrase once an episode. Nevertheless, Eh! Steve is vastly more popular and a much more recognizable symbol of the show.
- Teen Girl Squad is another good example.
- Strong Bad Lampshades this, along with The Other Darrin in the email original. After the departure of "Original Bubs," his temporary replacement "Onion Bubs" became a fan favorite.
- Onion Bubs! ONION BUUUUUBS!!!
- Played with in-universe in Strong Bad's commentary for an old King of Town toon. He hates every single thing about it except for the Blacksmith.Strong Bad: Oh, now this guy had some class. The Blacksmith, that guy was a good guy. I hung out with him after the cartoon; yeah, we had some good times...
- Red vs. Blue: Considering how the series is already an Ensemble Cast, there's numerous examples.
- Caboose, for whom the writers originally had no real plans other than for him to be a catalyst to annoy Church. Once he devolved into The Ditz of the group, he became very popular with the fandom, however, thanks to having some of the most hilarious dialogue in the series.
- Captain Butch Flowers was originally just a one shot character who dies after getting an aspirin shot, before appearing again as a minor (mind controlled) villain, and then again in a flashback sequence, where it's revealed he was a Freelancer specifically sent to guard Alpha and keep him a secret. Fans liked him even more after Season 14 showed him to be a hilariously dark sociopath who remains unfailingly polite and cheerful even when orchestrating the events of The Blood Gulch Chronicles and having his subordinates callously murdered when they're of no more use to him.
- Among the original Blood Gulch Crew, there's Lopez, the Spanish-speaking robotic marine/mechanic that serves as both the Beleaguered Assistant and Only Sane Man on the Red Team. Aside from just how strange he is as a concept, fans love him for his hilariously sarcastic and fatalistic mindset - Amidst all the strangeness of the series (intergalactic intrigue, war and drama, family tragedy, and more), Lopez couldn't care less and despises everyone while trying to avoid becoming just a head again.
- There's also Kaikaina Grif/"Sister" and Junior, Grif's younger sister and Tucker's son respectively. They were introduced in the midst of Season 5, and sadly didn't get to do a hell of a lot and have largely been Put on a Bus ever since aside from a few cameos. Fans love them both, but for rather (obviously) different reasons: Sister's beloved for not just being surprisingly friendly deep-down, but for being Grif's Morality Pet, endearingly ditzy, and having dialogue that constantly Crosses the Line Twice. Meanwhile, Junior's well liked for being surprisingly cute, helping show Tucker's softer side, and for introducing numerous storylines related to the Covenant (who don't really appear that much in Red vs. Blue).
- From what you can tell by the amount of views for 6 of the Speed Paints for Fairy Foxes done by creator StickFreeks herself, Kai, Kiki, and Louie might be in that territory.
- Cuddles may be the Series Mascot of Happy Tree Friends but Flippy and Flaky are undeniably the most popular characters. Unsurprisingly they're the Fan-Preferred Couple of the franchise.
- Greg the Spider from Arby 'n' the Chief.
- Velvet Scarlatina showed up in a single scene designed to show the audience the prejudice Faunus face on Remnant. She was such a huge hit with the fandom that she was fleshed out into a proper character, put on a team and given a recurring role in the show. Her combat outfit was even given an artists competition where fans could present their ideas, the winner being the design that was eventually used (albeit with a few modifications).
- The chainsaw-wielding White Fang heavy who kicked Weiss' ass in "No Brakes" has been getting a surprisingly large amount of mileage.
- If the Emperor Had a Text-to-Speech Device: Rogal Dorn, Fountain of Memes extraordinaire. His portrayal as The Comically Serious, entirely oblivious of the universe's insanity and utterly immune to others' snark (of which there is plenty) has made him beloved by the fanbase, to the point every single thing he says gets memetic status.
- Missi from The Vampair is likable for her charming looks, her vindictive personality, and her singing voice.
Ensemble Darkhorse / Web Animation