He said today that TfL was "always happy" to talk to private hire operators, and he was happy to meet anybody, but thought it was improper for politicians to interfere "with a quasi-judicial matter".
"What you can't do is have a situation where unfair pressure is brought on a quasi-judicial body," he said on BBC Radio 4.
"I appreciate that Uber has an army of PR experts. I appreciate that Uber has an army of lawyers; they've also made aggressive threats about taking us to court and all the rest of it," Khan added.
The mayor said the tech giant couldn't have it both ways; on the one hand acting in an aggressive manner, and on the other briefing journalists that they want to do a deal with TfL.
It was announced on Friday that Tfl had removed Uber's licence, saying the firm was "not fit and proper to hold a private hire operator licence".
And yesterday, Uber said it was seeking talks with the regulator in a bid to regain its licence to operate in the capital, as there has not yet been a meeting between them this year.
"We’re always willing to talk to Transport for London and the mayor. While we haven’t been asked to make any changes, we’d like to know what we can do. But that requires a dialogue we sadly haven’t been able to have recently,” Uber London general manager Tom Elvidge said.
“The 3.5m Londoners and 40,000 drivers who rely on our app would expect us to sit down and work together to get this right,” he added.
The Uber decision has faced criticism, with London business groups saying it was a damaging call for the capital.
Sam Dumitriu, research economist at the Adam Smith Institute, said it also "jeopardises the livelihoods of 40,000 drivers who choose to use the app because it gives them valuable flexibility".
But Khan said today he was clear that while London should be a place for new technology, and new firms to establish themselves, "but you've got to play by the rules".