Wnt11 and bone morphogenetic protein 2 (BMP-2) are key signaling factors for stem cell differentiation into functional cardiomyocytes (CMs). In this study, we elucidate the biological effect of BMP-2 and Wnt11 on bone marrow mesenchymal stromal cells (BM-MSCs) that differentiate into myocardial-like cells in a simulated myocardial microenvironment in vitro. A cell co-culture system was established with recombinant Wnt11 treatment of NIH/3T3 cells and CMs. BMP-2 was added in a diverse schedule to induce cardiomyogenic differentiation of BM-MSCs co-cultured under various conditions. The levels of cardiac-specific markers Nkx2.5, α-myosin heavy chain ( α-MHC), β-myosin heavy chain ( β-MHC) and cardiac troponin I (cTnI) were determined by reverse transcriptase polymerase chain reaction and immunocytochemistry to evaluate cardiomyogenic differentiation. Wnt11 or BMP-2 used on their own to differentiate BM-MSCs resulted in no expression of α-MHC and cTnI. Wnt11 alone in a myocardial microenvironment enhanced cardiomyogenic differentiation. BMP-2 demonstrated a dose-dependent effect on BM-MSC differentiation into myocardial-like cells. Addition of BMP to BM-MSCs at various time points resulted in varying effects on cardiomyogenic differentiation. The combination of Wnt11 and BMP-2 treatment in a temporal manner significantly enhanced cardiomyogenic differentiation of BM-MSCs, with high expressions of α-MHC, β-MHC, Nkx2.5 and cTnI upon co-culture with CMs. Our study demonstrates that the combination of Wnt11 and BMP-2 effectively promotes cardiomyogenic differentiation of BM-MSCs in vitro. The synergistic effect of Wnt11 and BMP-2 on the cardiomyogenic differentiation of BM-MSCs is further enhanced in a myocardial microenvironment.