Williams' hopes of playing in Saturday's doubles looked to have improved after their match was added to the order of play on Friday.
Their tie, against Colombia's Camila Osorio and Argentina's Solana Sierra, was not allocated a court and not due to be played before 16:30 BST.
It suggested Williams was being given as long as possible to declare herself fit.
Williams showed little sign of a problem during a three-set loss to 20-year-old Joint and did not ask for treatment. But the knee was said to have swelled quickly after she came off court.
The looseness of Saturday's plan also left Wimbledon with no scheduling issues in case the American superstars did withdraw.
Williams was due to practise at the All England Club at 16:00 but announced her decision to pull out about 15 minutes before.
The Williams sisters have won 12 singles titles between them, plus six doubles titles together, and the mood around the SW19 grounds was largely one of sadness and disappointment when it was announced the iconic pair would not play.
In her post announcing the decision, Serena Williams shared photographs of her wearing bandaging on the left knee and of what she said were syringes used to drain fluid from the area.
"I'm especially grateful to tournament director Jamie Baker and the entire tournament team for giving me every opportunity to play here," said Williams.
"Thank you to the fans after your incredible support and for making this comeback so meaningful.
"All I can say is stay tuned to a city near you."
Williams has not confirmed what her plans for the next stage of her comeback will be.
After Wimbledon, the season switches to the North American hard court swing which culminates at the US Open between 30 August and 13 September.
Williams, who turns 45 in September, is a six-time singles champion at her home Grand Slam tournament and played what many expected to be the final match of her career at Flushing Meadows in 2022.
The pain felt by Williams at being denied another opportunity to play with her big sister Venus, who has just turned 46, was laid bare in her announcement.
Given she has shown a constant refusal to accept defeat throughout her career, Williams will be annoyed she lost in the singles and could not play the doubles.
But the feeling in the Williams camp is that her comeback to Grand Slam tennis after four years away can still be regarded as a success.
Returning to singles was a bold move.
After just two doubles matches at Queen's and Berlin, Williams left the decision to play in the Wimbledon singles until as late as possible and even said in her pre-tournament news conference - with a laugh - she still wasn't completely sure why she had decided to do it.
There were glimpses of vintage Williams in a gallant defeat by Joint - particularly booming first serves and powerful winners - and her insatiable competitive spirit remains undimmed.
But there was also lots of rust. Her footwork and movement looked untidy - although that might have been down to the knee issue in the latter stages.
Overall, Williams and her team have been given enough encouragement from the All England Club that she will be able to win matches in the coming months.
The big question mark is whether she is physically equipped to do so.
Is she committed to continue putting in the hard work on the practice courts - which she needs to do to be competitive in the North American swing - and can her body stand up to the rigours of competitive matches?
