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Human stem cells are typically obtained for clinical application of cell therapy by isolating and subculturing adherent cells. The expansion of stem cells depends on the culture conditions and requires medium supplemented with fetal bovine serum (FBS) to generate relevant numbers of cells. However, the presence of FBS is a major obstacle for clinical use of the stem cells. Therefore, we have evaluated the capacity of expansion of amniotic fluid-derived stem cells (AFSCs) in a commercial serum-free medium in comparison with a conventional FBS-containing medium. AFSCs were expanded in two different culture media. Proliferation rate of the cells was higher in serum-free medium than in FBS-containing medium. Stem cells isolated in the both media were morphologically similar, and they were expressed identical surface antigens and mRNA of stem cell markers. AFSCs in the both media were also able to differentiate into the myogenic cells. These results suggest that a commercial serum-free medium (MesemCult-XF Medium) is an optimal medium for expansion of AFSCs. It allows a better cell expansion, preserves cell characteristics, and reduces the culture period. This medium seems suitable for clinical scale expansion of AFSCs.