Gates' exit from America First Policies came after the Associated Press reported this week that Manafort had sought to further Russian government interests in his work for a Russian businessman. Gates did not return CNN's requests for comment.
One administration source familiar with the matter called the departure "amicable." A second source told CNN that Gates was asked to leave after his name popped back into the news this week due to his connections to Manafort.
Gates previously served as Manafort's deputy when he was campaign chairman, and has long worked with him in the private sector, including in consulting work for foreign interests.
America First Policies confirmed the news Thursday on Twitter, tweeting: "Rick Gates is grateful @POTUS is standing up to #fakenews and supports agenda. @americanfirstpol is setup and strong- he's on to more ventures!"
Gates was one of six former Trump campaign aides who joined forces after the campaign to form the nonprofit after Trump was elected president in November. Gates was one of the group's top officials alongside the Trump campaign's digital director Brad Parscale and longtime Mike Pence aide Nick Ayers, both of whom remain at the helm of the nonprofit.
The Manafort ally's departure adds to the woes the nonprofit has faced in its first months.
Despite initially expecting to draw millions of dollars in funding from the wealthy family of Bob and Rebekah Mercer, America First Policies has floundered in its opening months amid squabbles between Rebekah Mercer and Parscale. The Mercers were among the top donors supporting Trump's presidential bid and maintain a close relationship with Trump advisers in the White House.
The nonprofit has not yet aired a single ad to boost Trump's health care push, the biggest legislative test of his presidency so far and an effort where pro-Trump outside groups had been expected to play a major role.
A spokesman for the group did not respond Thursday when asked if America First Policies planned to advertise for the bill.
Other group aides, beyond Gates, have even considered aiding multiple rival nonprofits at the same time, which would be a highly unusual relationship.
The Mercer family has yet to publicly commit to supporting the group.
CORRECTION: This story's headline has been updated to correctly identify America First Policies.