Members of the GATA-4, -5, and -6 subfamily of transcription factors are co-expressed with the homeoprotein Nkx 2.5 in the precardiac mesoderm during the earliest stages of its specification and are known to be important determinants of cardiac gene expression. Ample evidence suggests that GATA factors and Nkx 2.5 cross-regulate each other's expression; however, the temporal order of the expression of these transcription factors in vivo remains unresolved, and thus precise definition of the role of the products of the genes they transcribe in early development has been difficult to assess. We employed P19 CL6 mouse embryonic carcinoma cells as a model to investigate this problem, because these cells, like embryonic stem cells, can be induced to differentiate along multiple lineages. Here we demonstrate that when P19 CL6 cells are induced to differentiate to a cardiogenic lineage, the expression of GATA-4 and GATA-6 is up-regulated prior to the transcriptional activation of Nkx 2.5. Moreover, over-expression of GATA-4 or -6 at the time of Nkx 2.5 induction results in a significant up-regulation of endogenous Nkx 2.5 transcription. Finally, it is known that a Nkx-dependent enhancer is necessary for GATA-6 expression within cardiomyocytes of the developing mouse embryo. We demonstrate that within undifferentiated P19 CL6 cells, GATA-6 expression is subject to active repression by a novel upstream element that possesses binding sites for factors involved in transcriptional repression that are conserved between mammalian species.