Thymocytes undergo apoptosis during negative selection in vivo and following treatment with glucocorticoids or DNA-damaging drugs in vitro. The post-mitochondrial biochemical steps leading to apoptosis induced by these stimuli are well characterized, however, much less is known about the pathways connecting receptor triggering, apical caspase activation and induction of mitochondrial dysfunction. These stimuli specifically activate the kinase Cdk2 and this step is obligatory for these forms of thymocyte apoptosis. We report here that Cdk2 activation is a very early step during thymocyte apoptosis preceding apical caspase activation and phosphatidylserine exposure. Furthermore, Cdk2 activation is required for mitochondrial permeability disruption, cytochrome c release and, as a consequence, activation of the downstream caspases 9 and 3. Our data allow an integrated linear pathway regulating DNA damage and glucocorticoid-induced thymocyte apoptosis to be proposed.