The role of Fas ligand in T cell development and apoptosis in vivo has not been defined. The murine Fas ligand cDNA was placed under the control of a TCR CT promoter and enhancer minigene. Six lines of Fas ligand transgenic mice have been established. T cells from thymocyte, lymph node and spleen expressed increased levels of Fas ligand mRNA and high levels of biologically active Fas ligand determined by apoptosis of Fas-sensitive A20 target cells. There is increased apoptosis m vivo determined by TUNEL staining and a decreased population of CD4+CD8+ thymocytes and CD4+ thymocytes and spleen cells. Thymocytes and spleen T cells become sensitive to Fas-mediated apoptosis after activation. Mice were stimulated m vivo with anti-CD3 (100 meg/day) for three days and sacrificed one day later. This resulted in greatly increased thymocyte and spleen cell apoptosis determined by TUNEL staining and apoptosis of CD4+CD8+ thymocytes and CD4+ thymocytes and spleen cells. Surviving thymocyte and spleen cells in non-transgenic mice expressed low levels or no Fas, indicating T cell apoptosis after in vivo activation by antiCD3 is primarily mediated by Fas/Fas ligand. In Fas ligand transgenic mice, there was a discreet population of surviving cells that express high levels of Fas, indicating that a novel population of Fas apoptosis-resistant cells developed in these mice. We have previously reported that the Fas signaling defect is one mechanism for regulation of Fas apoptosis. The present results indicate that high levels of Fas ligand can result in both Fas-mediated apoptosis and in production of cells that are resistant to Fas apoptosis. Fas apoptosis resistant cells may play a critical role in autoimmunity, graft rejection, and in some hematopoietic malignancies.