A large number of thymocytes undergo apoptosis and die during maturation. Most thymocytes express high levels of Fas-antigen (CD95), however the role of the Fas/Fas-L system in mediating apoptosis of developing thymocytes has not been determined. Therefore we studied anti-Fas induced apoptosis in fetal thymic organ culture (FTOC) of C57BL/6" +/+ and C57BL/6-lpr/lpr mice. EM and TUNEL analysis indicated that large numbers of thymocytes undergo apoptosis primarily as cortical clusters on gestational day 16 in vivo and a decreasing apoptosis rate on the following days. Time-lapsed photography using confocal microscopy to analyze culture chambers containing FTOC revealed that the clearance of apoptotic bodies occurred rapidly and most of them were visible inside macrophages at gestational day 17. Fetal thymi of Ipr/lpr mice generated more thymocytes during early development compared to +/+ mice, which appeared to be due to decreased apoptosis in lpr/lpr mice. Immature newly generated CD4+8+ thymocytes of +/+ but not Ipr/lpr mice that expressed low levels of CDSrtCR complexes were highly sensitive to Fas mediated apoptosis in FTOC. However more mature CD4+8+ thymocytes that expressed high levels of CD3/TCR complexes were resistant to anti-Fas induced apoptosis. The engagement of low avidity CD3/TCR on Fas sensitive CD4+8+ thymocytes rescued them from Fas mediated apoptosis. The results indicate that most thymocytes undergo apoptosis during fetal thymic development when rearrangement of the TCR occurs and a signal through TCR/CD3 prevents thymocytes undergoing Fas mediated apoptosis. Our results suggest that Fas might play a critical role in mediating apoptosis in developing thymocytes that do not receive a survival signal through TCR/CD3.