Boris Johnson's First Deputy Mayor, Tim Parker, has quit - just over a month after taking up the post.
Mr Parker, 52, stood down after being told he would not be handed the role of running Transport for London (TfL) as had been previously agreed.
But the mayor's office said the former boss of Clarks shoes and the AA would stay on as an adviser.
Mr Parker is the third senior official to step down from Mr Johnson's administration in three months.
In July, Ray Lewis resigned two months into his post as deputy mayor for youth amid claims of financial irregularities.
A month earlier, James McGrath stepped down as director of political strategy at City Hall.
A further four deputy mayors are currently in office at City Hall.
When Mr Parker's appointment was announced in May, he agreed to become chairman of TfL as well as chief executive of London's governing body, the Greater London Authority.
But on Tuesday Mr Johnson said he would continue to chair the board of TfL, the local government body responsible for London's transport network.
He said this was "to ensure appropriate democratic accountability to the people of London".
In response, Mr Parker said: "I have concluded... that it would not be appropriate for an unelected official to chair a body which is responsible for most of the money and a large part of the brief of an elected mayor."
To lose one adviser is unfortunate, to lose two is careless but to lose three in four months shows the wheels are coming off this administration
Mike Tuffrey, Liberal Democrat leader
He said his position as an advisor "does not justify my full-time and exclusive commitment to the Greater London Authority, or the title of first deputy mayor".
"We have therefore decided to adjust the management structure and abolish that position," Mr Parker said.
Mr Johnson had been due to hand over to Mr Parker next month but decided his personal involvement in TfL was "crucial to being an effective mayor".
He said: "Over the last few weeks... it has become increasingly apparent to both of us that the nature of the decisions that need to be taken are highly political and there is no substitute for me, as the directly elected mayor, being in charge.
"There are limits, therefore, to what can be delegated."
Commenting on the latest resignation, leader of the London Liberal Democrat party, Mike Tuffrey said: "To lose one adviser is unfortunate, to lose two is careless but to lose three in four months shows the wheels are coming off this new administration.
"Why is Boris losing yet another adviser? Has Tim Parker discovered that running London isn't as easy as running private business?"
In a statement, the London Assembly Labour Group said the mayor was now in an "unsustainable position".
It said: "He needs to find some competent deputies to share his responsibilities; instead it looks like they are fighting like rats in a sack."
Tim Parker was due to take over as Chair of the Board for Transport for London
This page is best viewed in an up-to-date web browser with style sheets (CSS) enabled. While you will be able to view the content of this page in your current browser, you will not be able to get the full visual experience. Please consider upgrading your browser software or enabling style sheets (CSS) if you are able to do so.