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Objective:To investigate the feasibility of bone marrow stromal cells (BMSCs) differentiating into cardiomyocyte-like cells in heterogeneous cardiomyocytes microenvironment in vitro.Methods:Mouse GFP-BMSCs were isolated by centrifugation through a Ficoll step gradient and purified by plating culture and depletion of the non-adherent cells. Neonatal at cardiomyocytes (CMs) were isolated by enzymatic dissociation from hearts of 1-to 2-day-old Sprague-Dawley (SD) rats and differentially plated to remove fibroblasts. Mouse GFP-BMSCs were cocultured with neonatal rat CMs through direct and indirect contact, respectively. Cardiomyogenic differentiation of BMSCs was evaluated by immunostaining with nti-α-actin monoclonal antibody and observing synchronous contraction with adjacent CMs by phase contrast fluorescent contracting cells (rat-derived CMs) showed myotube-like formation and started to contract synchronously with adjacent cardiomyocytes. About 10% of the fluorescent GFP-BMSCs were cardiomyocyte-like cells as determined by cell morphology and positive actin staining.Conclusion:Direct cell-to-cell interaction with CMs is crucial for cardiomyogenic differentiation of BMSCs in heterogeneous CMs microenvironment in vitro. This provides a novel inducing pathway for directional differentiation of cardiovascular tissue engineering seed cells.