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Stem cells are cells characterized by their ability to differentiate into multiple types of cells. This unique property has the potential to lead to many promising solutions to human disease treatment. The stem cell differentiation process can be influenced by many factors. One of the important factors is the mechanical stimulation by modulating the extracellular matrix (ECM) elasticity. Stem cell stiffness increases as it differentiates to lower potency stem cells. This might be due to the reorganization of the cell cytoskeleton and could be confirmed by different imaging techniques. Atomic Force Microscopy (AFM) has been an ideal instrument in nanoscale imaging and mechanical property characterization because of the nature of its measurement. In this study, we applied the AFM with nanomanipulation capability to investigate mechanical properties of stem cells with and without differentiation in a time-lapse fashion. The experimental results showed that we were able to forecast the differentiation of mouse embryonic stem cells (mESC) around 24 hours after removal of Leukemia inhibitory factor (LIF). The stiffness of mESC after differentiation shows a one-fold increase over that of non-differentiated cells. Therefore the nanomechanical marker can be used as an early indicator for mESC differentiation.