This study investigates how geometric cues influence the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. The research demonstrates that cell shape, independent of soluble factors, significantly affects the differentiation of MSCs into adipocytes or osteoblasts. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and shapes with pentagonal symmetry but different subcellular curvature—each occupying the same area—show different adipogenesis and osteogenesis profiles. The results indicate that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment, verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular-related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. The study shows that subtle geometric cues can significantly influence the differentiation of MSCs by modulating mechanochemical signals and paracrine/autocrine factors. The research used microcontact printing to pattern the shapes of individual cells on a substrate, allowing for precise control over cell shape and differentiation. Cells cultured on different shapes showed varying degrees of osteogenesis and adipogenesis, with shapes that promoted increased contractility leading to preferential osteogenesis. The study also found that cytoskeletal manipulation, such as using pharmacological agents that disrupt the cytoskeleton, can remove the influence of shape on differentiation. RNA expression analysis revealed that cells on star shapes showed higher expression of osteogenic transcripts compared to cells on flower shapes. The study also identified the role of Wnt signaling and MAPK pathways in promoting osteogenesis, with contractile cells being more susceptible to soluble factors including Wnt signals. The findings suggest that geometric cues can play a significant role in directing the differentiation of MSCs, highlighting the importance of physical cues in stem cell fate determination.This study investigates how geometric cues influence the differentiation of human mesenchymal stem cells (MSCs) from bone marrow. The research demonstrates that cell shape, independent of soluble factors, significantly affects the differentiation of MSCs into adipocytes or osteoblasts. When exposed to competing soluble differentiation signals, cells cultured in rectangles with increasing aspect ratio and shapes with pentagonal symmetry but different subcellular curvature—each occupying the same area—show different adipogenesis and osteogenesis profiles. The results indicate that geometric features that increase actomyosin contractility promote osteogenesis and are consistent with in vivo characteristics of the microenvironment of the differentiated cells. Cytoskeletal-disrupting pharmacological agents modulate shape-based trends in lineage commitment, verifying the critical role of focal adhesion and myosin-generated contractility during differentiation. Microarray analysis and pathway inhibition studies suggest that contractile cells promote osteogenesis by enhancing c-Jun N-terminal kinase (JNK) and extracellular-related kinase (ERK1/2) activation in conjunction with elevated wingless-type (Wnt) signaling. The study shows that subtle geometric cues can significantly influence the differentiation of MSCs by modulating mechanochemical signals and paracrine/autocrine factors. The research used microcontact printing to pattern the shapes of individual cells on a substrate, allowing for precise control over cell shape and differentiation. Cells cultured on different shapes showed varying degrees of osteogenesis and adipogenesis, with shapes that promoted increased contractility leading to preferential osteogenesis. The study also found that cytoskeletal manipulation, such as using pharmacological agents that disrupt the cytoskeleton, can remove the influence of shape on differentiation. RNA expression analysis revealed that cells on star shapes showed higher expression of osteogenic transcripts compared to cells on flower shapes. The study also identified the role of Wnt signaling and MAPK pathways in promoting osteogenesis, with contractile cells being more susceptible to soluble factors including Wnt signals. The findings suggest that geometric cues can play a significant role in directing the differentiation of MSCs, highlighting the importance of physical cues in stem cell fate determination.