Background Our previous study showed the 150 mg/mL fetal cardiac supernatant (FCS) could induce differentiation of BMSCs into cardiomyocye-like cells without cardiomyocyte touch,but differentiation efficiency is not high enough.Inhibition of glycogen synthase kinase-3 enhanced the proliferation and survives of stem cells.We tested if 6-bromoindirubin-3-oxime (BIO,glycogen synthase kinase-3 inhibitor) enhances the effects of FCS on differentiation of BMSCs and explore the growth factors in FCS.Methods BMSCs were isolated from the femur and tibia of four-week-old male Sprague-Dawley rats and co-cultured with FCS (150 mg/mL) that was made from fetal hearts from nineteen-day pregnant Wistar rats.BIO with different concentration (0,1,10,and 100 nM) was introduced in culture dishes.Transforming growth factor beta 1 (TGF-β1),bone morphogenetic protein 2 (BMP-2) and Akt in cardiac supernatant and culture medium were assayed with ELISA methods.Results After co-culturing with FCS,beating myotubes were observed in 25.9 % BMSCs dishes after 1 to 2 weeks’ culture.The levels of TGF-β1 and BMP-2 in FCS concentrations were no more than that in young and adult cardiac supernatant.All BIO groups significantly enhanced the effects of FCS on differentiation of BMSCs into the cardiomyocyte-like cells (1 nM,83 %;10 nM,73 %;100 nM,100 %).Akt levels were higher in BMSCs cultural medium with FCS.Conclusions FCS could induce the differentiation of BMSCs into the cardiomyocyte-like cells.TGF-β1 and BMP-2 might not play a role in the differentiation of BMSCs induced by FCS.BIO enhanced the effects of FCS on the differentiation of BMSCs into cardiomyocyte-like cells,which might involve the Akt pathway. Background Our previous study showed the 150 mg / mL fetal cardiac supernatant (FCS) could induce differentiation of BMSCs into cardiomyocye-like cells without cardiomyocyte touch, but differentiation efficiency is not high enough. Inhibition of glycogen synthase kinase-3 enhanced the proliferation and survives of stem cells. We tested if 6-bromoindirubin-3-oxime (BIO, glycogen synthase kinase-3 inhibitor) enhances the effects of FCS on differentiation of BMSCs and explore the growth factors in FCS. Methods BMSCs were isolated from the femur and tibia of four-week old male Sprague-Dawley rats and co-cultured with FCS (150 mg / mL) that was made from fetal hearts from nineteen-day pregnant pregnant Wistar rats. BIO with different concentration (0,1,10, and 100 nM) was introduced in culture dishes. Transforming growth factor beta 1 (TGF-β1), bone morphogenetic protein 2 (BMP-2) and Akt in cardiac supernatant and culture medium were assayed with ELISA methods. Results After co-culturing with FCS, beating myotub es were observed in 25.9% of BMSCs dishes after 1 to 2 weeks’ culture. These levels of TGF-β1 and BMP-2 in FCS concentrations were no more than that in young and adult cardiac supernatant. All BIO groups significantly enhanced the effects of FCS on differentiation of BMSCs into the cardiomyocyte-like cells (1 nM, 83%; 10 nM, 73%; 100 nM, 100%). Akt levels were higher in BMSCs cultural medium with FCS.Conclusions FCS could induce the differentiation of BMSCs into the cardiomyocyte-like cells.TGF-β1 and BMP-2 might not play a role in the differentiation of BMSCs induced by FCS.BIO enhanced the effects of FCS on the differentiation of BMSCs into cardiomyocyte-like cells, which might involve the Akt pathway .