ObjectiveTo review the relative researches about mechanical stimulation of stem cells differentiation in stem cells microenvironment in vitro. MethodsThe recent related literature about stem cells differentiation in vitro was reviewed and summarized. ResultsThe mechanical loads (including shear stress, mechanical strain, and stress), substrates stiffness, substrates nanotopography, and cell shape were the 4 important aspects of mechanical factors regulating stem cells differentiation. The mechanical stimulation can simulate the in vivo microenvironment, which can alter the size, shape, alignment, and differentiation state of stem cells, can change the expression of their differentiation markers, and can affect the lineage commitment of stem cells. ConclusionMechanical stimulation play an important role in regulating stem cells differentiation and cells morphology in addition to chemical and biological factors.