Angry Birds: Black bird (choleric), white bird (melancholic), red bird (phlegmatic), and yellow bird (sanguine).
The four spirits of the Book of Prophecy in Avalon Code can be identified as this: Rempo (choleric), Ur (melancholic), Neaki (phlegmatic), and Mieli (sanguine).
In Blaze Union, the first five party members: Garlot (choleric), Jenon (melancholic), Medoute (phlegmatic), Siskier (sanguine), and Aegina (leukine). And after the party dynamics suffer an upheaval, things shake down like so: Gulcasa and Leon (choleric), Nessiah (melancholic), Baldus (phlegmatic), Emilia (sanguine), and Aegina is... still leukine.
Borderlands: Mordecai (melancholic), Lilith (phlegmatic), Roland (choleric), Brick (sanguine)
Borderlands 2: Axton (sanguine with a bit of choleric), Maya (melancholic), Salvador (choleric), Zer0 (phlegmatic). With DLC, there's also Gaige and Krieg, who are Sanguine and Choleric respectively.
Hyperion: Handsome Jack (choleric), Mr. Blake (melancholic), Professor Nakayama (sanguine), Angel (phlegmatic)
Catherine, in terms of Vincent and his three bros that he hangs out with at the Stray Sheep. Orlando (choleric), Jonny (melancholic), Vincent (phlegmatic), and Toby (sanguine).
Melancholic: Hinata, Koizumi, Pekoyama and Gundam.
Phlegmatic: Komaeda, Nanami and Sonia.
The four playable characters of Drakengard, though at times they muddle about with their humors, tend to fall into distinct ones: Arioch (sanguine), Caim (choleric), Leonard (melancholic), and Seere (phlegmatic).
Fate/stay night has Rin/Archer (choleric), Shirou/Saber (melancholic), Sakura (phlegmatic), and Rider (leukine).
Final Fantasy V is the definition of this trope, right down to the characters representing the same elements for each of the 4 crystals. Bartz (wind — sanguine), Faris (fire — choleric), Cara (earth — melancholic), and Lenna (water — phlegmatic).
Final Fantasy IX's first party: Steiner (choleric), Vivi (melancholic), Garnet (phlegmatic), and Zidane (sanguine).
Front Mission: The members of Carrion Crow seem to fit an ordinary Ensemble and a Combo Ensemble, two each, rather neatly:
Alder and Gentz (choleric), Commander Lloyd/custom name and Paul (melancholic), Hans and Bobby (phlegmatic), Peewee and Porunga (sanguine), and Frederick and Ralph (leukine).
Keith and Gregorio (sanguine/choleric), Natalie and Maury/Molly (melancholic/choleric), J.J. and Yeehin (melancholic/phlegmatic), and Sakata and Yang Meihua (sanguine/phlegmatic).
Gears of War's Delta Squad are like that: Marcus Fenix (choleric), Damon Baird (melancholic), Dominic Santiago (phlegmatic), and Augustus Cole (sanguine).
Grand Theft Auto IV: The four main characters fit: Brucie (choleric), Niko (melancholic), Little Jacob (phlegmatic), and Roman (sanguine).
The female leads of the visual novel G-Senjou no Maou are a picture-perfect example: Haru (choleric), Mizuha (melancholic), Tsubaki (phlegmatic), and Kanon (sanguine).
The playable characters of Kirby's Return to Dream Land. King Dedede (choleric), Meta Knight (melancholic), and Bandana Dee (phlegmatic), and Kirby (sanguine).
Due to the author being interested with the subject, MARDEK RPG does this. The author believes everyone has a primary and a secondary temperament, though. Perhaps the clearest ingame example is Legion: a construct created by an inventor. It was animated with dark magic, with the inventor saying the incantation - due to his stuttering, though, four souls entered the body instead of one - one for each temperament.
Mass Effect 2 has the starting four. Shepard (choleric), Miranda (melancholic), Jacob (phlegmatic), and Joker (sanguine).
Mass Effect 1 and Mass Effect 3 squads are often comprised this way: Shepard (choleric), Garrus (melancholic), Liara (phlegmatic), and Tali (phlegmatic). Likewise, Ashley (sanguine) and Kaiden (neutral phlegmatic).
Mega Man (Classic series): Forte/Bass (choleric), Proto Man/Blues (melancholic), Mega/Rock Man (phlegmatic), and Duo (sanguine).
Mega Man Battle Network: Lan and Gutsman.EXE (choleric), Mayl and Glyde.EXE (melancholic), Megaman.EXE (phlegmatic), Yai and Roll.EXE (sanguine), and Dex (leukine).
And later on, Dex is choleric (like his Navi); Chaud is melancholic (like his Navi); Mayl is phlegmatic (and so is Megaman.Exe, still); Yai is sanguine (like her Navi, still); and Lan is leukine.
Mega Man X: The sensitive, conscientious X (melancholic), Zero who does what X can't (choleric), the polite and fatherly Signas (phlegmatic), and the adventurous wise-guy Axl (sanguine).
Among the villains: Vile has nothing but choleric faults (and yikes!!); Lumine is phlegmatic; Dynamo is sanguine; and Sigma is leukine.
X-Hunters, counting Vile as the solo X-Hunter in his own right: Vile (choleric), Agile (melancholic), Serges (phlegmatic), and Violen (sanguine).
Capcom never made clear whether classic Mega/Rock Man is the same Mega Man as X. If they are the same, Mega Man underwent a major temperament change. note Some may argue that the melancholic temperament simply got brought to light more clearly as Mega Man got infused with human feelings.
Among the Four Guardians: The self-centered but playful Leviathan (choleric), the polite-but-confrontational Phantom (melancholic), the reliable and civil Harpuia (phlegmatic), and the encouraging but Trigger Happy Fefnir (sanguine).
Of the melancholic hero X a duplicate was made, who turned out more choleric than melancholic and not even remotely mature, scarcely one thing like the original X.
Evil trio of Zero 3: With what little personality he's shown to have, Omega seems to be phlegmatic/sanguine (opposite Copy-X); Weil seems to be leukine.
The Beauty and the Beast unit in Metal Gear Solid 4 applies: Raging Raven (choleric), Crying Wolf (melancholic), Screaming Mantis (phlegmatic), and Laughing Octopus (sanguine).
Nazi Zombies: Tank (choleric), Takeo (melancholic), Nikolai (phlegmatic), and Richtofen (sanguine).
Nier features has something of this as well. Nier (choleric), Emil (melancholic), Grimoire Weisse (phlegmatic), and Kaine (sanguine).
Judging by their programmed natures, the Pac-Man ghosts: Pinky is choleric (chases and ambushes); Inky is melancholic (cautious); Clyde is phlegmatic/leukine (indifferent and random); and Blinky is sanguine (cocky, always chases).
Intentionally invoked in Planescape: Torment. In the end game, you meet up with three copies of yourself, who are the good incarnation (melancholic), crazy incarnation (sanguine) and pragmatic incarnation (choleric). The character of the protagonist depends on how you've been playing him, but he does start out as phlegmatic.
Star Wars Republic Commando: The four Clone Commandos of Delta Squad are this. Delta-38, "Boss", is choleric, showing good leadership instincts. Delta-07, "Sev", is melancholic, being the Cold Sniper of the group and showing great care to place each round for maximum effenciency. Delta-40, "Fixer", is phlegmatic, being, as Taun We put it, "A pure and uncomplicated soldier." Delta-62, "Scorch", is sanguine, showing much enjoyment in blowing the CIS forces apart.
In Street Fighter II, the four members of Shadaloo: Balrog (choleric), Sagat (melancholic), M. Bison (phlegmatic), and Vega (sanguine).
In the Super Mario Bros. series and its Wario Land spinoff, the Mario Brothers and their parallels can form this schema: Wario (choleric), Mario (melancholic, but not brooding or depressive), Waluigi (phlegmatic), and Luigi (sanguine).
The ATX Team in Super Robot Wars. At first the lineup is Sanger (choleric), Bullet (melancholic), Kyosuke (phlegmatic), and Excellen (sanguine). Later, Kusuha was drafted into the team as the leukine. Later on, Sanger leaves, and Kyosuke takes on the choleric mantle; and when Lamia enters, she takes on the phlegmatic mantle.
The Colour Tuesday: Alex is the hotheaded driver of the story (choleric). Kyle is the determined and selfless (melancholic). Bella is a slightly airy Cloudcuckoolander (sanguine). Chadd, the last permanent cast member, is a born follower (phlegmatic).
The Team Fortress 2 classes can be seen throughout the spectrum: Scout (choleric), Soldier (sanguine-choleric), Pyro (sanguine), Demoman (choleric-melancholic), Heavy (leukine, leaning choleric), Engineer (phlegmatic), Medic (phlegmatic-sanguine), Sniper (melancholic), Spy (melancholic-phlegmatic)