The Bcl-2 relative Bak is thought to drive apoptosis by forming homo-oligomers that permeabilize mitochondria, but how it is activated and oligomerizes is unclear. To clarify these pivotal steps toward apoptosis, we have characterized multiple random loss-of-function Bak mutants and explored the mechanism of Bak conformation change during apoptosis. Single missense mutations located to the alpha helix 2-5 region of Bak, with most altering the BH3 domain or hydrophobic groove (BH1 domain). Loss of function invariably corresponded to impaired ability to oligomerize. An essential early step in Bak activation was shown to be exposure of the BH3 domain, which became reburied in dimers. We demonstrate that oligomerization involves insertion of the BH3 domain of one Bak molecule into the groove of another and may produce symmetric Bak dimers. We conclude that this BH3:groove interaction is essential to nucleate Bak oligomerization, which in turn is required for its proapoptotic function.