Microenvironment plays a critical role in directing the progression of stem cells into differentiated cells. It is necessary to investigate the role of cardiac microenvironment in directing the differentiation of adipose tissue-derived stem cells (ADSCs). Human ADSCs were cocultured with rat cardiomyocytes directly or indirectly by cell culture inserts. For direct coculture, hADSCs were labeled with carboxyfluorescein succinimidyl ester (CFSE), then mixed with cardiomyocytes at a 1∶5 ratio in complete media and seeded at a cell density of 50 000 cells/ml. Fluorescence-activated cell sorting was used to extract and examine the directly cocultured differentiated ADSCs. For indirect coculture, differentiated ADSCs were collected directly. The assays used include scanning electron microscope(SEM) and transmission electron microscope(TEM) for the ultrastructure of differentiated cells, immunostaining against myosin heavy chain, troponinⅠ and connecxin43, Western blotting and RT-PCR for the expression of cardiac specific proteins and genes respectively. Results showed that the differentiated ADSCs experienced the coculture presented cardiac ultrastructure and expressed cardiac specific genes and proteins, and the fractions of ADSCs expressing these markers by direct coculture were higher than those of indirect coculture. These data indicate that in addition to soluble signaling molecules, the direct cell-to-cell contact is obligatory in relaying the external cues of the microenvironment controlling the differentiation of ADSCs to cardiomyocytes.