The multiple departures came amid a surge in speculation about Blair's future following a newspaper report that he will resign next July.
Top officials also sent strong signals that Blair intended to leave office within a year.
But Blair warned the rebels that infighting would jeopardise the governing Labour Party's effort to hold on to power.
Tom Watson, a junior defence minister, said it was not in the interest of the country for Blair to remain in office.
At least six parliamentary private secretaries - unpaid ministerial aides - also resigned to protest against Blair's determination to stay in office.
They said they had quit rather than remove their names from a letter demanding that the prime minister step aside.
The letter said that while they supported the centrist direction Blair had taken the party, he was no longer the right man to lead it.
"Sadly, it is clear to us - as it is to almost the entire party and the entire country - that without an urgent change in the leadership of the party it becomes less likely that we will win [the next] election," expected in 2009, said the letter, released on Wednesday.
Watson was one of 17 Labour parliamentarians who signed the letter.
Blair responded by saying he had been planning to sack Watson anyway, describing him as "disloyal, discourteous and wrong" in signing the letter.
It is widely expected that Gordon Brown, the finance minister, will take over the leadership of both the Labour party and the country when Blair decides to step down.
Many Labour parliamentarians are becoming increasingly uneasy with Blair's refusal to set a date for his departure, after his 2004 announcement that he would not seek a fourth term in office.
Blair loyalists, however, believe that setting a date would signal a lengthy power struggle that could paralyse the government.
Blair, 53, winner of a record three consecutive elections for Labour, has seen his popularity dive after a series of government scandals over sleaze and mismanagement, as well as controversy over wars in Iraq and Lebanon.
Opinion polls put Labour well behind the opposition Conservatives, who have been revived by their new leader, David Cameron.