The SNP leader will face a vote next week following his decision to cut ties with his power-sharing partners in the Green Party.
The Scottish Green Party will join with rival MSPs to back a no-confidence motion in First Minister Humza Yousaf next week, after the SNP kicked its coalition partners out of government.
Mr Yousaf announced his plan to cut ties with the Greens earlier on Thursday, following a bitter row over the SNP’s climbdown on climate targets.
And soon after, the Scottish Conservatives announced it would lodge a vote of no-confidence in him, claiming the first minister had “failed” in his role and had “focused on the wrong priorities for Scotland”.
Both Labour and the Liberal Democrats agreed to back the motion, with its success hanging on whether Green Party MSPs joined the attack to give SNP critics a majority in Holyrood.
Its co-leaders Patrick Harvie and Lorna Slater have now confirmed their party they will join forces to condemn Mr Yousaf’s leadership, with Green sources telling Sky News there was serious anger among the ousted party.
How Mr Yousaf’s ex-SNP leadership rival could be crucial in vote
In the Scottish parliament, the SNP has 63 seats out of 129, two short of an outright majority.
The Greens have seven, the Conservatives 31, Labour has 22, the Liberal Democrats have four, there is one Alba MSP Ash Regan – an ex-SNP leadership rival of Mr Yousaf’s – and there is also presiding officer Alison Johnstone who is both an MSP and Scotland’s equivalent of the Commons speaker.
If Ms Regan, who was formerly part of the SNP but defected to Alex Salmond’s Alba Party last October, backs Mr Yousaf then that would mean both sides having 64 votes, and Ms Johnstone would be expected to vote in favour of the status quo, so the first minister would survive.
But if Ms Regan votes against Mr Yousaf, then the opposition parties will have 65 votes against the SNP’s 63, and the first minister would lose.
If the no-confidence vote passes, it will still be up to Mr Yousaf to decide on how to respond. However, it puts increasing pressure on his position if he fails to hold the confidence of the majority of the parliament.
However, if a no-confidence vote was passed on the government, the SNP administration would have to resign and appoint a new first minister within 28 days or call an election.
Speaking at a news conference, Mr Harvie said: “Humza Yousaf becoming first minister was on the basis of a political cooperation which both parties members signed in good faith, which Humza Yousaf endorsed, and even two days ago was still endorsing.
“He’s now chosen to end that. That’s his decision and it can’t come without consequences.”
Both he and Ms Slater denied that supporting the no-confidence vote was “revenge”.
Mr Harvie added: “This is about how we achieve the greatest political change for Scotland.
“Humza Yousaf has decided to abandon the vehicle that was delivering that progressive change for Scotland. We think that’s a profound mistake.”
The power-sharing deal between the SNP and the Greens was made in 2021, after Nicola Sturgeon’s party came in just shy of an outright majority in the Holyrood election of the same year.