Fremantle star Nat Fyfe and St Kilda's Jack Billings are out of the AFLX competition as teams continue to focus on youngsters for the new format.
The Dockers originally named Fyfe in their squad for Thursday's first six-team tournament in Adelaide, but he was pulled out early on Tuesday morning.
After fronting the media at last week's AFLX launch, Billings will not play on Friday night at Etihad Stadium because of a hamstring injury.
Melbourne's squad for the six-team tournament at Etihad Stadium features Tomas Bugg, who has not played in the senior side since his six-game suspension last season for striking Callum Mills.
Christian Petracca and Tom McDonald are also in the Demons' squad.
North Melbourne best and fairest winner Shaun Higgins headlines their lineup for Friday night, with Ben Cunnington and Jarrad Waite also in the lineup.
Essendon's squad includes Gold Coast recruit Adam Saad, plus rising star winner Andrew McGrath, AFLX captain David Zaharakis and speedster Anthony McDonald-Tipungwuti.
Premiership star Liam Shiels will captain Hawthorn, who also picked Jack Gunston, Luke Breust, Ryan Burton and James Sicily.
While Billings is out of action for the Saints, Seb Ross and Luke Dunstan are in their squad.
The Saints will also unveil top-10 draft picks Hunter Clark and Nick Coffield in Friday night's two matches.
Five top-10 picks from last year's national draft will play on Friday night, with Carlton naming Paddy Dow and Lachie O'Brien and North including Luke Davies-Uniacke.