Abstract Micropatterns of arginine–glycine–aspartic acid (RGD) on poly(ethylene glycol) (PEG) hydrogels were fabricated. Under an appropriate size of microislands on this strong and persistent non-fouling background, single mesenchymal stem cells (MSCs) from rats were well localized, keeping the same adhesive area but different shapes. The cell shapes influenced the differentiation of MSCs, and the osteogenic and adipogenic differentiations exhibited different trends. According to comparison between square and rectangular cells, optimal adipogenic differentiation occurred at aspect ratio (AR) 1, but the optimal osteogenic differentiation was found when AR was about 2. We further interpreted the optimal ratios as reflecting the inherent global anisotropy of free adipoblasts and osteoblasts on unpatterned culture plates. According to comparison between globally isotropic circular, square, triangular, and star cells, the optimal adipogenic and osteogenic differentiations happened in circular and star cells, respectively. In this case we found that extents of both adipogenic and osteogenic differentiations were linearly related to cell perimeter, which reflects the non-roundness or local anisotropy of cells. Hence, the present study makes semi-quantitative investigations of the effects of cell shape on differentiation of stem cells based on a material technique, and reveals that the shape anisotropy is very important in directing the lineage commitments of stem cells.