E2F transcriptional activity controls the expression of many of the genes required for G₁ to S phase progression. E2F1, one member of the E2F family, plays an important role in the induction of apoptosis. We have examined the role of the E2F1 transcription factor in apoptosis during T-cell maturation in the thymus. We show that E2F1 is required for the apoptosis of autoimmune immature T cells during thymic negative selection in vivo. This T-cell receptor-mediated apoptosis coincides with the E2F1-dependent increase of p19-ARF mRNA and p53 protein levels. In contrast, E2F1 is not required for the induction of apoptosis by glucocorticoids or DNA damage. These results demonstrate a specific role for E2F1, which triggers a pathway leading to ARF and p53 induction, in a physiological apoptosis pathway that is uncoupled from a normal proliferative event.