The proximal promoter of lck directs gene expression exclusively in T cells. To investigate the developmental regulation of the lck proximal promoter activity and its relationship to T cell lineage commitment, a green fluorescence protein (GFP) transgenic (Tg) mouse in which the GFP expression is under the control of the proximal promoter of lck was created. In the adult GFP-Tg mice, >90% of CD4⁺CD8⁺ and CD4⁺CD8^– thymocytes, and the majority of CD4^–CD8⁺ and CD4^–CD8^– [double-negative (DN)] thymocytes were highly positive for GFP. Slightly lower but substantial levels of expression of GFP was also observed in mature splenic T cells. No GFP⁺ cells was detected in non-T lineage subsets, including mature and immature B cells, CD5⁺ B cells, and NK cells, indicating a preserved tissue specificity of the promoter. The earliest GFP⁺ cells detected were found in the CD44⁺CD25^– DN thymocyte subpopulation. The developmental potential of GFP^– and GFP⁺ cells in the CD44⁺CD25^– DN fraction was examined using in vitro culture systems. The generation of substantial numbers of αβ and γδ T cells as well as NK cells was demonstrated from both GFP^– and GFP⁺ cells. However, no development of B cells or dendritic cells was detected from GFP⁺ CD44⁺CD25^– DN thymocytes. These results suggest that the progenitors expressing lck proximal promoter activity in the CD44⁺CD25^– DN thymocyte subset have lost most of the progenitor potential for the B and dendritic cell lineage. Thus, progression of T cell lineage restriction in the earliest thymic population can be visualized by lck proximal promoter activity, suggesting a potential role of Lck in the T cell lineage commitment.