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Masahiro Sakurai stated that the Villager from Animal Crossing was removed from Brawl during the planning stages because "he wasn't suited for battle".
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Attachment The track "Castle / Boss Fortress (Super Mario World / SMB 3)" was originally going to be in the Delfino Plaza stage, but was later moved to the Luigi's Mansion stage instead.
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Attachment There is unused tile for the Stage Builder which can't be used normally. It resembles a bump, and is fully functional.
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An unused graphic of the Mario icon from Super Smash Bros. Melee can be found in the game's files. The Mario icon could mean that all the icons were going to be reused from Melee, but it also could have simply been a placeholder graphic.
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The only three English voice actors from Star Fox Assault's western dub to reprise their respective roles in Super Smash Bros. Brawl's Lylat Cruise conversations were:

•Jim Walker as Fox McCloud
•Alesia Glidewell as Krystal
•Mike McAuliffe as Slippy Toad

All the other characters were recasted:

•Falco Lombardi, now voiced by Dex Manley (originally Mike Madeoy)
•Wolf O'Donnell, now voiced by Jay Ward (originally Grant Goodeve)
•Peppy Hare, also now voiced by Dex Manley (originally Henry Dardenne)
•Leon Powalski, now voiced by Jim Walker (originally David Scully)
•Panther Caroso, now voiced by Eric Newsome (also originally David Scully)

This is not the case in the Japanese version of Super Smash Bros. Brawl, where literally of the Japanese voice cast from Assault, that also heavily comprised of returning voice actors from Star Fox 64, reprised their roles. More specifically, Kenji Nojima, Hisao Egawa, Kyoko Tongu, Tomohisa Aso, Teiya Ichiryusai, Mahito Ōba, Shinobu Satouchi and Tetsu Inada reprising their roles Fox, Falco, Peppy, Slippy, Krystal, Wolf, Leon and Panther respectively.
person CuriousUserX90 calendar_month June 13, 2014
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Sakurai considered making Miis Playable, but decided against it. According to Sakurai, it didn't seem right at the time to have Miis punching and kicking. Miis were however added in the game's sequel six years later.
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A black Nintendo DS Lite can be found in the boxes on one of the backgrounds in diorama mode.
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In Super Smash Bros. Brawl, Sonic's victory theme is actually the "Stage Clear" theme from Sonic the Hedgehog 2006, making it the only victory fanfare taken directly from another game.
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There is an extensive amount of music that is listed within the game's data, but the tracks themselves are not present in the final version.
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Attachment An unused HUD element named "InfAdvLevel0000" was apparently intended to display a shrinking bar for 50 frames (5/6 of a second) with a "level number" in a circle. The label "pp" is even more mysterious. The black areas of the textures would have been transparent in-game.
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During the Subspace Emissary, Ike will shout "Great Aether!" before attacking the Ancient Minister in a cutscene. However, the attack he does is merely a normal Aether.

This may have been done to match the pacing of the Japanese version, where he says "ten" as he leaps into the air, and "kuu" as he swings his sword down.
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There are multiple glitches that can occur if a character begins their Final Smash as the bridge is reforming in the stage 'Bridge of Eldin". The most notable of these is that R.O.B. will enter his final smash, Diffusion Beam, and stay in that state for the rest of the match, no matter how long. He will also remain invincible.
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With enough damage, it is possible to "destroy" a Landmaster, forcing the final smash to end early. However, without hacking, it is extremely difficult to pull off.
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An unused subaction and corresponding animation exist for Zero Suit Samus' Plasma Whip Side-B, labelled "SpecialSItemGet". There are also two other unused throwing animations for Samus labeled as "SpecialSThrowS" and "SpecialSThrowHi".

Presumably, the attack would originally have grabbed items from a distance and slung them away as a projectile, a function that would have been unique to the character. It may have been removed due to it interfering when players were attempting to hit another player with the whip.
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Attachment An unused character select screen exists in the game for a wi-fi mode named Slipspace. This mode is limited to "With Anyone", and unlike regular wi-fi modes, there is no space on the screen for additional players to join. In the settings bar, the player can set the number of minutes for the mode.

How this mode would have worked and whether it is operational in the game is unknown. The description text for the mode is:
"Set your time and leap into the battle slipspace!"
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Attachment Wolf has an unused attack animation which looks similar to that of Fox's rapid kick.
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The stage "Green Hill Zone" has a large amount of extra geometry outside of the camera view, a fact that the game's director, Masahiro Sakurai, noted on the Smash Bros. Dojo when the stage was revealed.
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Attachment An unused character select screen menu exists in the game for a wi-fi version of Boss Battles. It is unknown if this mode is functional and why it goes unused.

The description text for the mode reads:
"Take on 10 bosses with the help of a friend! (2 players)"
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Attachment Kirby has an unused animation that appears to be him walking with a hammer and then swinging it with full force in the same manner as King Dedede's Jet Hammer move. One Smash-focused Nintendo Direct in April 2014 included footage of Kirby charging up and releasing his hammer, in Super Smash Bros. for Wii U
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Fox and Falco both have unused animations that appear to be their blasters misfiring.
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