Fetal thymus organ culture system (FTOC), a well-known model used for the study of TCRαβ development, was employed to study TCRγδ cell development. It was found that different waves of TCRγδ cells develop from precursors within the fetal thymi at the time in vitro culture. Subsets of fetal thymocytes were analyzed by flow cytometry and 2-D gel biochemical analysis was performed. After 5 days in FTOC, Vγ3+ and Vγ2+ cells were dominant. By day 12 FTOC, the absolute number of Vγ3+ cells decreased while Vγ2+ and Vγ4+ cells became dominant. These observations suggest that the thymic micro-environment affects the thymic waves of TCRγδ subsets. Furthermore, the effect of TCR/antigen interaction in the development of TCRγδ cells was examined with anti-TCR mAbs added into the FTOC. Anti-CD3 mAb added to day 5 and day 12 FTOC inhibited TCRγδ development, especially Vγ4+ cells. On the other hand, Vγ2+ cells were relatively resistant to the addition of anti-TCR mAb. The reduction of TCRγδ+ thymocytes was not due to the modulation of TCR molecules and could be reversed by Cyclosporin A (CsA). These results suggest that TCR ligation negatively regulates the development of TCRγδ cells in a Vγ-specific manner.