In the present study we addressed the function of the transcriptional activator Zrf1 in the generation of the 3 germ layers during in vitro development. Currently, Zrf1 is rather regarded as a factor that drives the expression of neuronal genes. Here, we have employed mouse embryonic stem cells and P19 cells to understand the role of Zrf1 in the generation of mesoderm-derived tissues like adipocytes, cartilage and heart. Our data shows that Zrf1 is essential for the transcriptional activation of genes that give rise to mesoderm and in particular heart development. In both, the mESC and P19 systems, we provide evidence that Zrf1 contributes to the generation of functional cardiomyocytes. We further demonstrate that Zrf1 binds to the transcription start sites (TSSs) of heart tissue-specific genes from the first and second heart field where it drives their temporal expression during differentiation. Taken together, we have identified Zrf1 as a novel regulator of the mesodermal lineage that might facilitate spatiotemporal expression of genes.