ABSTRACTTranscriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood owing, in part, to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single-cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles.Mesp1, a transiently expressed mesodermal transcription factor, is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells inMesp1mutants, albeit mislocalized, prompting us to investigate the scope of the role ofMesp1in CPC emergence and differentiation.Mesp1mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and crucial cardiac transcription factors, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single-cell chromatin accessibility analysis defined aMesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results revealMesp1-independent aspects of early CPC specification and underscore aMesp1-dependent regulatory landscape required for progression through cardiogenesis.