They have been remanded in custody to appear at Southwark Crown Court at a later date.
Deputy Director Paul Foster, head of the NCA's National Cyber Crime Unit, said: "Today's charges are a key step in what has been a lengthy and complex investigation.
"This attack caused significant disruption and millions in losses to TfL, part of the UK's critical national infrastructure."
The court heard that the TfL hack occurred while Mr Flowers was on bail after being arrested in connection with multiple ransomware hacks.
After he was arrested, detectives found evidence he had also targeted US healthcare companies.
Mr Flowers has been charged with conspiring, with others, to infiltrate and damage the networks of SSM Health Care Corporation and attempting to do the same to Sutter Health.
When Mr Flowers appeared in court, he wore a grey hoodie with "off the grid" written on it. Mr Jubair sat next to him, wearing a black hoodie and black glasses.
Neither man spoke to each other during the proceedings.
The hack disrupted TfL services for three months.
TfL wrote to around 5,000 customers to say there may have been unauthorised access to their personal information such as bank account numbers and sort codes.
Data including names, emails and home addresses had been accessed, TfL added.
In a statement on Thursday afternoon, it said: "We welcome this announcement by the National Crime Agency that two people have now been charged in relation to the cyber incident which impacted our operations last year."
Earlier this year, the NCA warned of an increasing threat from cyber criminal gangs based in the UK and other English-speaking countries, such as Scattered Spider.
