In reply, McCullum hit a valiant 72 off 44 deliveries with seven sixes and two fours, while No. 7 batsman Ishan Kishan hit four sixes and two fours in his quickfire 39.
But Lions still lost by 21 runs as they finished at 192-7.
Leg-spinner Yuzvendra Chahal (3-31), who got McCullum in the 15th over, had earlier done the damage by taking the wickets of captain Suresh Raina and Dwayne Smith.
Gayle had the milestone on his mind in his 290th Twenty20 as he ran three singles off his first five deliveries to achieve the feat after Raina won the toss and opted to field.
The West Indian left-hander smashed seven towering sixes and five fours, putting on 122 runs for the opening wicket stand with Kohli off just 76 balls.
A huge score was always on cards after Gayle fell to Basil Thampi's perfect yorker in the 13th over.
Even in the absence of injured AB de Villiers, Royal Challengers had enough batting power as Kohli completed his second half century in the tournament but holed out at deep midwicket in the 16th over.
Jadhav ruined the bowling figures of Ravindra Jadeja (0-57) by smashing the left-arm spinner for 17 runs in the last over. It was the first time in T20s that Jadeja had conceded more than 50 runs in his four-over spell.
Australian medium fast bowler Andrew Tye, who achieved a hat trick last Friday against Rising Pune Supergiant, gave away 16 runs in his first three overs but conceded 18 off his last six deliveries as Jadhav belted two sixes and a four.
Royal Challengers now have four points from six matches and are sixth in the standings, with Lions in last place with a solitary victory and four losses.
On Wednesday, defending champions Sunrisers Hyderabad will host Delhi Daredevils.
