Yorkshire, chasing 184 to win a first Blast Roses game away from home since 2014, looked on course after Brook (72) and Tom Kohler-Cadmore (67) shared 115 for the fourth wicket.
But fast bowler Richard Gleeson trapped the in-form Brook, who has been called into the England Test squad for the first time, lbw with only one run needed to send the 14,000 crowd wild.
Ollie Pope scored 62 from 46 balls as Surrey defeated Glamorgan by four wickets with one ball to spare in front of 16,000 at the Kia Oval.
Pope was joined by Sam Curran in a fourth-wicket stand of 62 and Surrey got home at 174 for six despite a late flurry of wickets as Michael Hogan almost pulled the game out of the hat for the visitors.
A half-century from Joe Clarke and 47 by Tom Moores saw Nottinghamshire launch their campaign with a tight four-wicket win over Worcestershire at Trent Bridge.
Seamer Luke Fletcher took a career-best five for 33 as the Rapids were restricted to 164 for nine in their 20 overs before Clarke hit 52 from 34 balls and Moores clubbed three sixes in his 31-ball assault as Nottinghamshire clinched victory with just two balls to spare.
James Bracey registered a career-best T20 score as Gloucestershire beat Sussex by 41 runs.
Without a win in any of their previous seven matches in all formats this season, Gloucestershire were indebted to their England international, who hit 70 from 45 balls to help the home side reach 193 for seven after being put in.
Sussex’s reply faltered after Mohammad Rizwan went for 58, with six wickets then falling for the addition of only 23 runs.
Chris Lynn put Durham to the sword with a display of brutal power hitting in Northamptonshire’s 31-run victory.
In his first home game for the Steelbacks in the competition, Lynn took an instant liking to Wantage Road as he blasted 83 from just 46 balls, sharing a 149-run partnership with Ben Curran.
Northants’ total of 223 for four from their 20 overs proved more than enough as Durham were bowled out for 192 after 18.5 overs.
Luke Hollman and Toby Roland-Jones bowled Middlesex to a 10-run victory over Hampshire, their second win in succession.
Leg-spinner Hollman claimed three for 34 while fast bowler Roland-Jones returned two for 19 as Middlesex defended 163 with relative ease.
Birmingham Bears survived a late Derbyshire surge to win an exciting game at Derby by three runs.
The Bears smashed 89 off the last five overs to reach 200 for the second night running but the Falcons fought back and came close to pulling off what would have been their biggest T20 run chase.
Brooke Guest’s 54 from 34 balls almost swung the game in a dramatic finale but Craig Miles kept his nerve as the Falcons finished on 197 for eight.
Essex claimed a four-wicket victory over Kent with two balls to spare in a tense derby at Canterbury. Paul Walter top-scored with an unbeaten 47 as the visitors reached 188 for six in reply to Kent’s 184 for four.