Adipose-derived stem cells (ASCs) constitute a promising source for cell therapy and tissue engineering approaches as they can be easily obtained in large quantities with minimal patient discomfort. Moreover, ASCs have multilineage differentiation capacity. Among these, differentiation capacity along the myogenic lineage is of particular interest since myogenic precursors are scarce and obtaining large number of cells from skeletal muscle biopsies are difficult. Muscle cells are multinucleated single cells and multinucleation, along with the expression of muscle specific proteins and transcription factors, is an important verification of myogenesis. In this study, the effect of culture conditions, namely the composition of myogenic induction medium, culture medium, cell seeding density and substrate stiffness were studied in order to enhance ASC myogenesis efficiency. Results showed that induced cells fused, formed multinucleated cells and expressed muscle specific protein desmin while the efficiency was higher when cultured on polyacrylamide gels mimicking muscle stiffness. On the other hand, no signal of desmin staining and multinucleation was observed in uninduced control cultures. It was found that induction with the cocktail composed of 10 mu M 5-Azacytidine, 5% horse serum and 1% fetal bovine serum maximizes ASC multinucleation. As multinucleated ASCs have great potential for use in muscle regenerative therapies, this study represents an important enhancement towards their clinical use.