The line-up for the 2015 event is yet to be revealed, but Radio 1 host Zane Lowe couldn’t resist cheekily asking Flo whether she was booked to play.
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Florence's new album 'How Big, How Blue, How Beautiful' is due out in June
Rather than answering, the singer-songwriter stayed silent before laughing, prompting fans on Twitter to speculate over whether Florence and her band will be heading to Worthy Farm.
“So Florence basically just confirmed Glastonbury,” wrote one fan, while another also speculated over the rest of the line-up: “Would comfortably put money on Florence headlining Glastonbury after her interview with Zane Lowe tonight. So… Foo's, Coldplay and Florence?”
Florence And the Machine last played at Glasto in 2010, a year after release of their debut album ‘Lungs’.
Despite achieving huge success, both in terms of critical acclaim and record sales, Florence took a year out after the second album ‘Ceremonials’.
See all the other acts rumoured to be heading to Glasto below...
Glastonbury 2015 Line-Up Rumours
(01 of13)
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Taylor Swift has a Glastonbury-sized gap in her '1989' World Tour schedule, and Michael Eavis has stated that he'd love the singer to perform at Worthy Farm. Watch this space... (credit:Greg Allen/Invision/AP)
(02 of13)
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AC/DC have already confirmed that they're working on a new album, before heading off on tour again - could a stop at Worthy Farm be on the cards? (credit:Scott Legato via Getty Images)
(03 of13)
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Plenty of Glastonbury fans are keeping their fingers crossed for a Fleetwood Mac annoucement. (credit:Chris Pizzello/Invision/AP)
(04 of13)
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Could Queen and Adam Lambert be heading for Somerset? Brian May has refused to confirm - or deny - the rumours... (credit:Greg Allen/Invision/AP)
(05 of13)
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Well, it wouldn't be Glastonbury without Fatboy Slim, would it? (credit:Jon Furniss/Invision/AP)
(06 of13)
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After weeks of speculation over a possible secret set at 2014's festival, Prince and his band didn't perform there. Could 2015 be the year? (credit:Suzan/Suzan)
(07 of13)
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Adele's famously said she'll never do festivals - could Michael Eavis convince her to change her mind? (credit:Ian West/PA Wire)
(08 of13)
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Could Florence's comeback include a stop at Glasto? (credit:John Davisson/Invision/AP)
(09 of13)
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Rockers Muse are being tipped by bookmakers. (credit:Sharon Latham/Manchester City FC)
(10 of13)
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If Oasis ever reunite, surely Glastonbury would be the perfect place to do it? (credit:ASSOCIATED PRESS)
(11 of13)
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Foo Fighters graced the Main Stage in 1998 so we think it's about time they returned. (credit:Jim Dyson via Getty Images)
(12 of13)
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Well, if Beyoncé can... (credit:Mark Davis/NBC via Getty Images)
(13 of13)
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Coldplay's seventh album is in the works, and rumour has it that Chris and his bandmates could be heading for the Pyramid Stage once again. (credit:Powers Imagery/Invision/AP)