GATA-3 is a zinc-finger transcription factor that is essential for both early T cell development and Th2 cell differentiation. To quantify GATA-3 expression during T cell development in vivo in the mouse, the GATA-3 gene was targeted by insertion of a lacZ reporter by homologous recombination in embryonic stem (ES) cells. Although we could detect GATA-3 + cells throughout T cell development in the thymus, the proportions of GATA-3 + cells varied considerably between the distinct differentiation stages. The two periods of TCR ยง and g gene recombination, which occur in quiescent or slowly dividing cells, were associated with low proportions of GATA-3 + cells. Conversely, the stage of rapidly proliferating cells, which insulates these two waves of TCR rearrangement, was characterized by a large proportion of GATA-3 + cells. In addition, we generated chimeric mice by injection of GATA-3-deficient, lacZ-expressing ES cells into wild-type blastocysts. In this in vivo competition analysis, no contribution of GATA-3-deficient cells to the T cell lineage was detected, not even in the earliest CD44 + CD25 -double-negative (CD4 -CD8 -) cell stage in the thymus. These results parallel data implicating other GATA family members as key regulators of proliferation and survival of early hematopoietic cells. We therefore propose that GATA-3 is required for the expansion of T cell progenitors, and for the control of subsequent proliferation steps, which alternate periods of TCR recombination in the thymus.