Human mesenchymal stem cells (hMSCs) have the potential to differentiate into numerous cell types in vivo and in vitro. The ability to control hMSCs lineage commitment would bring great benefits to regenerative medicine. Many efforts have been made to use biochemical method to regulate hMSCs growth, whereas recently more progress contributes to the effort of physical cues on hMSCs behavior. In this work, highly controlled substrate topography was utilized to influence cell behavior including adhesion, proliferation and differentiation. In the first part, hMSCs were cultured on femtosecond laser machined polymer substrates including three sizes of microchannels (width-interval-depth: 20-20-20, 30-30-20 and 80-30-20; µm). Cytoskeleton of hMSCs was better aligned and elongated along the narrow laser induced microchannels (20-20-20 and 30-30-20). Meanwhile, cells on these scaffold also showed significant up-regulation of several hallmark myogenic markers. It was also suggested that RhoA/ROCK signaling is involved in myogenesis of hMSCs on these scaffolds. Then, laser machining and electrospinning techniques were utilized to obtain a bi-layered hybrid scaffold with hierarchical topographical features. HMSCs were cultured in vitro in the bi-layered hybrid scaffolds, and cell orientation, proliferation and differentiation were evaluated. The results proved that, without appearance of bio-chemical factors, these hierarchical scaffolds could offer a conducive microenvironment for enhancing hMSCs proliferation, viability, orientation and myogenesis in vitro.