Stem cells have emerged as potentially useful cells for regenerative medicine applications. To fully harness this potential, it is important to develop in vitro cell culture platforms with spatially regulated mechanical, chemical, and biological cues to induce the differentiation of stem cells. In this study, a cell culture platform was constructed that used polydopamine (PDA)-coated parafilm. The modified parafilm supports cell attachment and proliferation. In addition, because of the superb plasticity and ductility of the parafilm, it can be easily micropatterned to regulate the spatial arrangements of cells, and can exert different mechanical tensions. Specifically, we constructed a PDA-coated parafilm with grooved micropatterns to induce the osteogenic differentiation of stem cells. Adipose-derived mesenchymal stem cells that were cultured on the PDA-coated parafilm exhibited significantly higher osteogenic commitment in response to mechanical and spatial cues compared to the ones without stretch. Our findings may open new opportunities for inducing osteogenesis of stem cells in vitro using the platform that combines mechanical and spatial cues.