Manchester United, Liverpool, Arsenal and Tottenham have followed Chelsea and Manchester City in pulling out from the proposed Super League.
City were the first Premier League side to confirm they were pulling out of the breakaway competition.
The Premier League leaders said in a statement on Tuesday evening: "Manchester City Football Club can confirm that it has formally enacted the procedures to withdraw from the group developing plans for a European Super League."
The rest of the clubs soon followed Man City and confirmed the news through press release.
The 12-team Super League was announced on Sunday to widespread condemnation.
Manchester City confirmed they have "formally enacted the procedures to withdraw" from the Super League.
Liverpool said their involvement in the proposed breakaway league "has been discontinued".
Manchester United said they had "listened carefully to the reaction from our fans, the UK government and other key stakeholders" in making their decision to not take part."
Arsenal apologised and said they had "made a mistake", adding they were withdrawing after listening to their fans and the "wider football community".
Tottenham chairman Daniel Levy said the club regretted the "anxiety and upset" caused by the proposal.
Chelsea, whose fans protested against the planned breakaway league outside Stamford Bridge ahead of Tuesday's Premier League match against Brighton, are understood to have changed their mind because of the overwhelming negative worldwide reaction.
The competition's announcement on Sunday, made by 12 founding clubs - including Manchester United, City, Liverpool, Chelsea, Arsenal and Tottenham from the Premier League - was met with widespread criticism.