Most thymocytes express high levels of Fas Ag (Apo-1/CD95); however, the role of Fas/Fas ligand-mediated apoptosis in thymocyte development remains unclear. During gestational development of thymocytes in C57BL/6(B6) +/+ mice, the highest levels of Fas ligand mRNA and Fas ligand protein expression were detected at gestational day (GD) 15, and there was a ninefold decrease in Fas ligand mRNA expression between GD 15 and 17 accompanied by a sixfold increase in Fas mRNA. Apoptotic thymocytes were first detected in the medulla at GD 15, and increasing numbers of cortical clusters and scattered, single apoptotic cells were present on GD 16 and 17. Thus, early apoptosis correlated with high expression of Fas ligand. High levels of Fas ligand mRNA were maintained throughout gestational development in thymocytes of Fas- deficient B6-lpr/lpr mice, but cortical clusters and scattered apoptotic cells were decreased relative to B6 +/+ mice before GD 17. Kinetic analysis of fetal thymic organ cultures treated with anti-Fas Ab demonstrated that thymocytes become sensitive to Fas-mediated apoptosis during the transition from the CD4-CD8- to the CD4+CD8+ phenotype. More mature CD4+CD8+ thymocytes and CD4+ and CD8+ thymocytes became resistant to Fas-mediated apoptosis after GD 17, despite high expression of Fas. However, low avidity engagement of the TCR on Fas-sensitive CD4+CD8+ thymocytes before GD 17 induced resistance to Fas-mediated apoptosis. The present results indicate that Fas plays a critical role in mediating apoptosis during early gestational thymocyte development and that thymocytes that receive a survival signal through TCR/CD3 become resistant to Fas-mediated apoptosis.