Expression of the stem cell antigen-1 (Sca1) denotes cardiac progenitors in the adult mouse heart and a clonogenic subset (CSP) is identified by the side population phenotype. Clonal CSP cells differentiate to cardiomyocyte, endothelial and smooth muscle lineages when grafted in vivo; however, the instructive signals directing adoption of these fates are unknown. Similarly, in vitro conditions for directed differentiation of CSP have yet to be defined. To facilitate high content screening approaches for the investigation of signals directing cardiomyocyte differentiation, a lentiviral fluorescent reporter assay based on the 5.5 kb Myh6 promoter was developed. In validation experiments comparing expression in a range of differentiated cell types, the Myh6-based construct demonstrated superior specificity for the cardiomyocyte fate than a TNNT2-based construct that was assessed as a potential alternative. However, in proof of concept, high content screens, Myh6 reporter gene induction was observed in the absence of endogenous Myh6 gene expression. Ectopic expression of lentiviral reporter constructs based on tissue specific promoter is reported elsewhere and these findings highlight the potential for false positive induction in screening applications. Unexpectedly, cardiomyocyte differentiation was not observed under any of the conditions tested, although smooth muscle differentiation occurred under conditions of low serum. The discrepancy between in vivo and in vitro lineage capacity suggests that the methods for differentiation investigated here were inadequate. Before phenotypic screens of cell fate can be instigated for CSP clones, further studies are required to better define which CSP clones are most cardiogenic and to establish methods for culture that better recapitulate the endogenous microenvironment.