Abstract: Cardiomyocytes from human iPS cells have promise in disease modeling, drug discovery, and therapy, but the challenge remains to create mature and organized cardiomyocytes like those of the native heart. While groups have increased maturity of iPS-derived cardiomycytes by extended culture and electrical, metabolic, and biomechanical stimulation, we hypothesized that epigenetic activation during the formation of cardiac progenitors could enhance their capacity to form mature cells. We found that administration of the innate immune agonist PolyIC during formation of cardiac progenitors in defined small molecule differentiation increased histone acetylation and decreased HDAC expression without increasing cell death. To test if epigenetic activation could restore signaling factors present in development, we measured the expression of endogenous Wnt ligands and inhibitors in cardiac differentiation lacking the small molecule inhibitor of Wnt and observed that PolyIC rescued endogenous Wnt inhibition and cardiomyocyte differentiation by augmenting decreased expression of Wnt 3 ligand. Compared to normal cardiac progenitors, activated progenitors had enhanced proliferation, future cardiomyocyte yield, and a dominant increase in cell proliferation pathways in RNAseq. This was lead by genes such as Jagged1 of the Notch pathway expressed in the developing cardiac crescent and heart tube, and Notch inhibition blocked progenitor proliferation. Activated progenitors differentiated into more mature cardiomyocytes based on larger size, optical upstroke velocity, oxidative metabolism, and expression of markers of CM maturation including cTnI, cardiac actin, and αMHC, which were blocked by Notch inhibition. Singularized activated progenitors could self-assemble and differentiate into organized, synchronized, beating cardiomyocyte sheets containing a network of HCN4+ conduction system cells that were sensitive to the nodal current blocker ivrabradine in contrast to the poorly coupled cardiomyocytes from nonactivated progenitors. Impact: Activated cardiac progenitors with the capacity to give rise to cardiomyocytes with enhanced maturation and organization hold promise for improving disease modeling, drug screening and therapy.