The zinc finger transcription factor GATA-1 is essential for both primitive (embryonic) and definitive (adult) erythropoiesis. To define the roles of GATA-1 in the production and differentiation of primitive and definitive erythrocytes, we established GATA-1-null embryonic stem cell lines in which GATA-1 was able to be conditionally expressed by using the tetracycline conditional gene expression system. The cells were subjected to hematopoietic differentiation by coculturing on OP9 stroma cells. We expressed GATA-1 in the course of primitive and definitive erythropoiesis and analyzed the ability of GATA-1 to rescue the defective erythropoiesis caused by the GATA-1 null mutation. Our results show that GATA-1 functions in the proliferation and maturation of erythrocytes in a distinctive manner. The early-stage expression of GATA-1 during both primitive and definitive erythropoiesis was sufficient to promote the proliferation of red blood cells. In contrast, the late-stage expression of GATA-1 was indispensable to the terminal differentiation of primitive and definitive erythrocytes. Thus, GATA-1 affects the proliferation and differentiation of erythrocytes by different mechanisms.