GATA-4 is a cardiac-specific member of the GATA family of zinc finger transcription factors. During embryogenesis, GATA-4 expression is detected very early in the cardiogenic area and persists later in the developing heart. Studies have shown that GATA-4 is a potent transcriptional activator of several cardiac muscle-specific genes and a key regulator of the cardiomyocyte gene program. Consistent with a role for GATA-4 in cardiomyocyte formation, inhibition of GATA-4 expression by antisense transcripts interferes with expression of cardiac muscle genes and blocks development of beating cardiomyocytes in P19 embryonic stem cells. In order to better define the function of GATA-4 in cardiogenesis, we have carried out molecular analysis of early stages of cardiomyocyte differentiation in GATA-4-deficient P19 cell lines and in P19 cells stably overexpressing GATA-4. The results indicate that GATA-4 is not required for either endodermal or mesodermal commitment or for initiation of the cardiac pathway. However, in the absence of GATA-4, differentiation is blocked at the precardiac (cardioblasts) stage and cells are lost through extensive apoptosis. In contrast, ectopic expression of GATA-4 in P19 cells accelerates cardiogenesis and markedly increases (over 10-fold) the number of terminally differentiated beating cardiomyocytes following cell aggregation. Together, these findings suggest that, in addition to its role in activation of the cardiac genetic program, GATA-4 may be the nuclear target of inductive and/or survival factors for precardiac cells.