While the visitors lead by a goal following the first leg in Florence, their hosts at De Grolsch Veste will be seeking to extend their first European adventure for eight years.
Match preview
Despite claiming a 2-1 win at Stadio Artemio Franchi last week's opening leg, Fiorentina may feel they let slip a chance to kill off the tie and effectively secure progress to the Conference League's group stage.
Vincenzo Italiano's men must travel to the Netherlands with the slimmest of advantages, after surging into a two-goal lead thanks to goals from Nicolas Gonzalez and Arthur Cabral but then conceding midway through the second half.
That Twente found the net with their only shot on target will sting the Serie A side all the more, but having posted a pair of wins and gone unbeaten in their first three matches of the new season, the signs are positive that they can continue their growth since Italiano's appointment last year.
The Viola made their return to continental competition for the first time in six years after finishing seventh in Italy's top flight last term; establishing themselves as one of the league's most entertaining teams.
Since the summer break, they seem to have lost little of their momentum, as they edged out promoted Cremonese 3-2 on the opening day, and then followed their Conference League win with a hard-fought goalless draw in a Tuscan derby last weekend.
Though they managed to keep a rare clean sheet, Fiorentina were unable to break down an Empoli side reduced to 10 men for the final quarter of the game, and their record on the road continues to contrast with some exceptional home form.
Now set to tackle their first - and possibly last - European trip of the season, the Viola may have won 15 of their last 21 matches at the Franchi, but are always more vulnerable away from Florence.
With Vaclav Cerny's 64th-minute strike last week giving them a crucial boost before Thursday's home leg, Twente may well be confident of causing an upset back in Enschede.
Having had the benefit of a weekend off from Eredivisie duty to prepare, Ron Jans will expect his side to operate at full tilt for Fiorentina's visit, and a promising start to the season suggests they should be up to the task.
Twente finished fourth last term, qualifying for a long-awaited return to Europe, and they breezed past Cukaricki in their first Conference League tie with a resounding 7-2 aggregate victory earlier this month.
During that time, the Tukkers also kicked off their league campaign by beating NEC Nijmegen, and then followed that up with a 3-0 home win over Fortuna Sittard.
Indeed, they had won each of their previous six matches before being beaten in Florence, so hopes of reaching a European group stage for the first time since their top-flight return in 2019 remain very much alive.
FC Twente Europa Conference League form:
FC Twente form (all competitions):
Fiorentina Europa Conference League form:
Fiorentina form (all competitions):
Team News
Last week, Vincenzo Italiano made six changes to his first starting XI of the Serie A season and still managed to produce a winning performance, and the Fiorentina coach is expected to switch things up again, given a growing squad.
Sofyan Amrabat put in an impressive display in a defensive midfield role and should be recalled on Thursday after being an unused substitute against Empoli.
First-leg scorers Nicolas Gonzalez and Arthur Cabral are likely to be brought back into the front three, while Luka Jovic drops to the bench; Pietro Terracciano returns in goal. Szymon Zurkowski and Igor could be sidelined, though, and Gaetano Castrovilli is a longer-term injury absentee.
Meanwhile, the hosts will again turn to Ricky van Wolfswinkel to lead the line up front, and the well-travelled 33-year-old will aim to make an impact on his return to Europe after scoring 16 times in the league last term and laying on eight assists.
Captain Wout Brama - holder of Twente's record for European appearances, with 66 to date - hopes to win a race for fitness after a groin injury kept him out of the first leg, while fellow midfielders Ramiz Zerrouki and Casper Staring recently returned from the treatment room and should feature at some stage.
FC Twente possible starting lineup:
Unnerstall; Brenet, Hilgers, Propper, Smal; Zerrouki, Sadilek; Cerny, Vlap, Misidjan; Van Wolfswinkel
Fiorentina possible starting lineup:
Terracciano; Venuti, Milenkovic, Nastasic, Biraghi; Duncan, Amrabat, Mandragora; Gonzalez, Cabral, Sottil