The present study is aimed to examine the shape effect on lineage commitment of stem cells in growth medium free of external chemical induction factors. Aspect ratios (ARs) of cells were controlled by micropatterns with cell-adhesive microislands of AR 1, 2 and 8 on the potent nonfouling background of poly(ethylene glycol) hydrogels, and the single stem cells were well shaped for 19 days. Mesenchymal stem cells (MSCs) derived from rat bone marrow were cultured in osteogenic medium, adipogenic medium, mixed coinduction medium, and also growth medium; alkaline phosphatase (ALP) and oil droplets were employed as indicators of osteoblasts and adipocytes, respectively. Those indicators were well observed in all of three induction media as early as day 7, and also in growth medium at a longer culture time till day 13. While a significant monotonic decrease of adipogenesis was observed with the increase of AR, a non-monotonic change of osteogenesis was found with optimal AR about 2. The relative gene expressions further verified the above findings. As a result, cell shape itself is an inherent cue to regulate stem cell differentiation, let alone with or without external chemical induction factors. Such a shape effect disappeared upon addition of a microfilament inhibitor cytochalasin D or a Rho-associated protein kinase (ROCK) inhibitor Y-27632. So, formation of cytoskeleton is necessary for the shape effect, and the ROCK-pathway-related cell tension is responsible for the shape effect on the lineage commitment of stem cells even in growth medium.