Seven-time grand slam singles winner Williams stormed through the first set without losing a game and had two break points to go 5-4 up in the second, but an inspired Flipkens turned things around to triumph 0-6 6-4 6-2.
"I wasn't expecting to play that well in the first set, so in the second set I just didn't handle it well - I was pretty close to closing that match out there," Williams said on www.wtatennis.com.
"Then I got down in the third. I think I just sprayed a few balls and got a little tentative. I definitely didn't make the best choices out there, but in general it was really fun to be back out there again, and I'm getting ready for Cincinnati next week."
Dominika Cibulkova of Slovakia sprung a surprise in her second-round clash with Angelique Kerber, ousting the eighth-seeded German 6-7 (0/7) 6-2 7-5.
"Angelique was putting everything back - her defensive game is really good, and I just had to be patient and aggressive at the same time," said Cibulkova, who fought from 4-1 down in the decider to prevail in two hours 57 minutes.
Third seed Agnieszka Radwanska of Poland had few problems in her second-round encounter, seeing off Belgium's Yanina Wickmayer 6-2 6-3.
In other first-round action, Magdalena Rybarikova followed up a successful title defence at the Citi Open in Washington by toppling Romanian world number 254 Alexandra Dulgheru 6-4 6-3.
Russian Svetlana Kuznetsova, the former French Open and US Open champion, suffered a surprising 3-6 7-5 7-5 opening-round loss to American 19-year-old Lauren Davis.
Maria Kirilenko, seeded 11, fared better than her compatriot and emerged a sharp 6-2 6-1 winner against Croatian Petra Martic.
Serbians Jelena Jankovic and Ana Ivanovic, 15th and 16th seeds respectively, took contrasting routes. Jankovic was a 7-6 (8/6) 4-6 6-3 winner against Georgian Anna Tatishvili, while Ivanovic breezed past Chinese Taipei's Hsieh Su-wei 6-1 6-2.
Australian 12th seed Samantha Stosur recovered well to get past Israel's Julia Glushko 5-7 6-2 6-3, while Francesca Schiavone of Italy also needed three sets but progressed with a 2-6 6-3 6-3 victory over South Africa's Chanelle Scheepers.
Sharon Fichman overcame fellow Canadian Stephanie Dubois 5-7 6-2 6-2, with another home player also advancing in the form of Eugenie Bouchard, who defeated Russia's Alisa Kleybanova 6-3 6-1.
And there were straight-sets wins for Carla Suarez-Navarro, Ekaterina Makarova and Alize Cornet against Jamie Hampton, Anastasia Rodionova and Elena Vesnina, respectively.