Spec Ops 1:

Recruit Mockingbird

Epic boss not required.

Spec Ops 2:

Recruit Emma Frost

Colossus, Scarlet Witch, and Phoenix required

Spec Ops 3:

Recruit Magik

Gambit, Nightcrawler, Rogue, and Black Panther required

Spec Ops 4:

Recruit Ghost Rider

Daredevil, Gambit, Hercules, and Quicksilver required

Spec Ops 5:

Recruit Valkyrie

Beast, Kitty Pryde, Storm, and Wolverine required

Spec Ops 6:

Recruit Havok

Beast, Deadpool, and Tigra required

Spec Ops 7:

Recruit Hank Pym

Cable, Spider-Man, and Wasp required

Spec Ops 8:

Recruit Vision

Black Knight, Captain Britain, and Psylocke required

Spec Ops 9:

Recruit Rescue

Punisher, Union Jack, and War Machine required

Spec Ops 10:

Recruit Wonder Man

Black Cat, Thundra, and X-23 required

Spec Ops 11:

Recruit Daimon Hellstrom

Fantomex, Iron Fist, and Scarlet Witch required

Spec Ops 12:

Recruit Ares

Hercules, Shatterstar, and Thor required

Spec Ops 13: 

Recruit Black Bolt

Psylocke, Red Hulk, and Wasp required

Spec Ops 14:

Recruit Heimdall

Mockingbird, She-Hulk, and Valkyrie required

Spec Ops 15:

Recruit Nico Minoru

Bishop, Spider-Man, Hawkeye, Black Cat, Black Widow, and Emma Frost required

Spec Ops 16:

Recruit Iceman

Havok, Colossus, and Spiral required

Spec Ops 17:

Recruit Falcon

Fandral, Punisher, and Red Hulk required

Spec Ops 18:

Recruit Anti-Venom

Agent Venom, Gamora, and Ms. Marvel required

Spec Ops 19:

Recruit Cannonball

Daimon Hellstrom, Nightcrawler, and Hogun required

Spec Ops 20:

Recruit Star-Lord

Rocket Raccoon, Ares, and Phoenix required

Spec Ops 21:

Recruit Ka-Zar

Drax, Groot, and Tigra required

Spec Ops 22:

Recruit Blade and Morbius

Medusa, Gorgon and Nico Minoru required

Spec Ops 23:

Recruit Crystal

Iceman, Molly Hayes, and Hercules required

In alphabetical order the used heroes are:

Agent Venom, Ares, 2x Beast, Bishop, 2x Black Cat, Black Knight, Black Panther, Black Widow, Cable, Captain Britain, 2x Colossus, Daimon Hellstrom, Daredevil, Deadpool, Drax, Emma Frost, Fandral, Fantomex, 2x Gambit, Gamora, Gorgon, Groot, Havok, Hawkeye, 3x Hercules, Hogun, Iceman, Iron Fist, Kitty Pryde, Medusa, Mockingbird, Molly Hayes, Ms. Marvel, Nico Minoru, 2x Nightcrawler, 2x Phoenix, 2x Psylocke, 2x Punisher, Quicksilver, 2x Red Hulk, Rocket Raccoon, Rogue, 2x Scarlet Witch, Shatterstar, She-Hulk, 2x Spider-Man, Spiral, Storm, Thor, Thundra, 2x Tigra, Union Jack, Valkyrie, War Machine, 2x Wasp, Wolverine, X-23

Haven't been required for Spec Ops yet:

Iron Man, Captain America, Cyclops, Dr. Strange, Hulk, Human Torch, Invisible Woman, Luke Cage, Mr. Fantastic, Sif, Spider-Woman, ThingMagikGhost RiderHank Pym, Vision, Rescue, Wonder Man, Angel, Black BoltDoctor VoodooDominoHeimdallFalcon, Anti-VenomCannonballStar-Lord, Ka-Zar, BladeKarnak, Crystal.

I've included a couple of Spec Ops heroes that aren't available for general recruitment yet in that list since they will be at some point but for now I've put their names in italics. 

As it is clear from the list above several heroes have been used multiple times and some have never been used. And then there's hero requirements for season 2 and various Covert Ops so you really can't predict what's next.

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