Objective To establish a technique for sensitively reflecting the micro-mechanical changes of human mesenchymal stem cells (hMSCs) in their early differentiation process by mechanical phenotype. Methods Based on atomic force microscopy (AFM), peak force quantitative nanomechanical mapping (PF-QNM) technique was used to measure the nanomechanical spectroscopy of hMSCs during differentiation induced by different concentrations of lithium chloride. Results The nanomechanical spectroscopy of hMSCs induced by 4 mmol/L and 30 mmol/L lithium chloride was significantly different after 48 h, while there was significant difference in Young's modulus after 72 h. Conclusion Nanomechanical spectroscopy is superior to Young's modulus in reflecting the change of mechanical properties of hMSCs in their early differentiation phase. Mechanical phenotype based on nanomechanical spectroscopy can be used as a physical biomarker to identify the hMSCs at their early differentiation stage.