South Down MLA John McCallister quit on Thursday night over the decision to field a unionist unity candidate in the upcoming Mid Ulster by-election.
"If he is resigning on principle, why did he not resign over the selection of an agreed candidate in Rodney Connor in Fermanagh & South Tyrone?" Mr Nesbitt asked on Friday.
Within hours, Mr McCallister was followed out of the party by Lagan Valley MLA Basil McCrea - and the pair could now form a new political party.
"I reject Basil's claim that, in my leadership campaign, I advocated 'no big idea'. What I said was there was no quick fix and there was no 'big idea' beyond a long, hard slog on the ground. Hard work was the big idea," Mr Nesbitt said.
Speaking about both former party members, the UUP leader said: "It is a matter of huge regret that, following a unanimous vote of the Mid-Ulster Ulster Unionist Constituency Association, some individuals chose to attack the party on the airwaves.
"The Party reviewed key policies in 2012 and neither John nor Basil attended internal meetings or contributed in any meaningful way. Indeed, John attended more Northern Ireland Conservative events over that period than he attended Ulster Unionist reviews."
My vision remains for a revitalised, pluralist, non-sectarian progressive political party remains undimmed. That party is the Ulster Unionist Party.
Mike Nesbitt, UUP
In announcing his resignation, Mr McCrea said the UUP had lost its way as party and that he did not know what it stood for anymore.
He told UTV: "The party made a decision last night to endorse a unity candidate - I had been on record both in public and actually in private [saying] that I thought this was the wrong way to go for the party, for the people of Northern Ireland, and indeed, for the electorate of mid- Ulster.
"And so, the party having made it's decision, I felt I could no longer remain in the Ulster Unionist Party."
He added that Unionist unity did not work.
"The challenge I put out to people is - if you can tell me what the UUP stands for that is different from the DUP, I'll be interested in what that is because I can't see it."
Mr McCrea confirmed that he and John McCallister are in talks about "the appropriate way forward."
He added he felt the forthcoming by-election in Mid-Ulster - where joint Unionist candidate Nigel Lutton, the son of a murdered RUC reservist, will contest the seat against Sinn Féin's Francie Molloy - would become a sectarian battle.
According to Mr McCrea, both he and Mr McCallister had made their decisions knowing that it would be very difficult to get re-elected - but they felt it was something they had to do.
Earlier this month, Mr McCrea attended an UUP disciplinary hearing after he criticised the party over its handling of issues linked to the Union flag dispute.
He received a formal warning, which stopped short of expulsion or suspension.
Mr McCrea had the UUP whip removed by Mike Nesbitt after he said he agreed with the Alliance Party motion of flying the flag on designated days at Belfast City Hall.
He said he felt his stance was in accordance with UUP policy, but later opposed his party over a DUP amendment to a UUP motion calling for the Assembly's commitment to inclusivity, mutual respect, peace and democracy, in the wake of the flag unrest.
The DUP wanted to remove a reference to the Good Friday Agreement.
Mr McCallister's resignation came in an open letter to the party leader, in which he said that he "fundamentally disagrees" with the principle of Unionist unity.
The former deputy leader of the party's Assembly group said the cooperation with the DUP was leading local politics "back into the sectarian trenches".
He also accused Mike Nesbitt of failing to lead unionism into an "alternative to the politics of sectarian headcounts".
Speaking about the new Mid-Ulster candidate, Mr McCallister said: "It's nothing personal to Nigel, I know Nigel. It is about principle. I am opposed to unionist unity and I do not want to be part of that."
UTV's political correspondent Ken Reid said that in the immediate future the UUP will only have 13 MLAs in the Assembly.
"It will put the party into fourth position, because the SDLP have 14. Although Assembly rules will mean that it will have no actual practical effect on the day to day running of the Assembly.
"One of the more interesting aspects is that Basil McCrea is saying that himself and John McCallister will be talking about the formation of a new party."
Independent MLA David McClarty told UTV that he expects to enter talks with the two men in the coming days and weeks.
Mr McClarty left the UUP in 2011. He has been an MLA for East Londonderry since 1998, and quit the party when he was deselected by them, becoming an independent politician.
He added: "Now had I been a member of the Ulster Unionist Party last night, I would have been sitting in the same [TV] studio as John McCallister saying the same things."
Mr McClarty said all three men wanted to take Northern Ireland away from the "tribal politics" of the past.