The study identifies isl1+ cardiac progenitors in the postnatal hearts of rats, mice, and humans. These progenitors, which are maintained in a pluripotent state by a cardiac mesenchymal feeder layer, can be selectively marked and purified using tamoxifen-inducible Cre/lox technology. Co-culture experiments with neonatal myocytes demonstrate that isl1+ cells efficiently differentiate into mature cardiomyocytes without cell fusion, exhibiting stable expression of myocytic markers, intact Ca2+-cycling, and the generation of action potentials. This discovery provides a genetically based system to study cardiac cell lineage formation and maturation, offering insights into congenital and adult cardiac diseases.The study identifies isl1+ cardiac progenitors in the postnatal hearts of rats, mice, and humans. These progenitors, which are maintained in a pluripotent state by a cardiac mesenchymal feeder layer, can be selectively marked and purified using tamoxifen-inducible Cre/lox technology. Co-culture experiments with neonatal myocytes demonstrate that isl1+ cells efficiently differentiate into mature cardiomyocytes without cell fusion, exhibiting stable expression of myocytic markers, intact Ca2+-cycling, and the generation of action potentials. This discovery provides a genetically based system to study cardiac cell lineage formation and maturation, offering insights into congenital and adult cardiac diseases.