Abstract Following bone marrow transplantation, patients often suffer from immune incompetence by reduced or late T cell development. Moreover, adult bone marrow stem cells have a lower capacity to generate T cells compared with fetal liver- and umbilical cord blood-derived progenitors. Therefore, enhancing thymic-dependent T cell generation might hold great therapeutic potential. GATA-3 is a transcription factor that is essential in T cell development. In this study we examined the therapeutic potential of GATA-3 to enhance T cell generation by overexpressing GATA-3 in T cell progenitors followed by fetal thymic organ culture (FTOC). We observed that early during FTOC, there was an enhanced differentiation toward the double positive stage of T cell development. From day 10 of FTOC, however, overexpression of GATA-3 induced a severe reduction in thymic cellularity, which probably correlates with the absence of a functional TCR-β chain. We further show that the frequency of apoptosis was increased in GATA-3-transduced thymocytes. Despite the absence of a functional TCR-β chain, GATA-3 transduced progenitors were able to differentiate into CD8β+ double positive thymocytes. This study shows that a strictly regulated expression of GATA-3 is essential for normal T cell development and this puts severe restrictions on the potential therapeutic use of continuously overexpressed GATA-3.