Recent studies have demonstrated that direct cell-to-cell interaction is one of the microenvironment factors for transdifferentiation of adult stem cells into cardiomyocytes. We investigated whether transdifferentiation of mesenchymal stem cells (MSCs) into cardiomyocytes was dependent on developmental stages of cocultured cardiomyocytes, and direct cell-to-cell interaction was essential for transdifferentiation. MSCs were isolated from adult rat and cocultured in four different ways: (1) with neonatal cardiomyocytes, (2) with adult cardiomyocytes, (3) with neonatal cardiomyocytes on the cell culture inserts, and (4) with the conditioned medium from neonatal cardiomyocytes. After 5 days of coculture with neonatal cardiomyocytes, 9.40±1.15% of 1,1′-dioctadecyl-1-3,3,3′,3′-tetramethylindocarbocyanine perchlorate labeled MSCs expressed sarcomeric-α-actinin. Immunocytochemistry showed that only these MSCs expressed the cardiac markers and were not observed with other coculture condition as well as conditioned medium. Calcein-AM labeling of cardiomyocytes showed gap junctional communication between 56.1±2.0% of MSCs (24 h after labeling, n=5) and neonatal cardiomyocytes. These findings suggest that MSCs are capable of differentiating into cardiomyocytes when directly cocultured with neonatal cardiomyocytes by cell-to-cell interaction, but not with adult cardiomyocytes or conditioned medium.