Apoptotic protease-activating factor 1 (Apaf-1) is a component of the apoptosome which is required for the activation of procaspase-9. As Apaf-1 knockout (KO) (Apaf-1 -/-) mice die before birth, the role of Apaf-1 during thymic selection was investigated using 5 day foetal thymic organ culture (FTOC) of thymi obtained at gestational day 15. There was a lower ratio of CD4 single-positive (SP) to CD8 SP cells and decreased apoptosis of CD4 +CD8 + (DP) thymocytes from Apaf-1 -/- mice compared with wild-type. To determine if these defects resulted in increased production of neglected thymocytes, the Apaf-1 -/- mice were crossed with the T-cell receptor (TCR)-α-chain KO mice. There was no difference in thymocyte development in the thymi of TCR-α -/- Apaf-1 -/- and TCR-α -/- Apaf-1 +/+ mice 5 days after FTOC. To determine if Apaf-1 is involved in apoptosis during death by negative or positive selection, FTOC of the thymus of Apaf-1 -/- D b/HY TCR-αβ transgenic (Tg) mice was carried out. There was decreased apoptosis of the HY clonal-specific M33 + thymocytes and an increased percentage of the autoreactive CD8 +M33 + thymocytes in male, but not female Apaf-1 -/- D b/HY TCR Tg mice. Our data suggest that Apaf-1 is not involved in positive selection or death by neglect, but may have a partial role in negative selection during early thymic T-cell development.