Stem cell behaviors are regulated by multiple microenvironmental cues. As an external signal, mechanical stiffness of the extracellular matrix is capable of governing stem cell fate determination, but how this biophysical cue is translated into intracellular signaling remains elusive. Here, we elucidate mechanisms by which stem cells respond to microenvironmental stiffness through the dynamics of the cytoskeletal network, leading to changes in gene expression via biophysical transduction signaling pathways in two-dimensional culture. Furthermore, a putative rapid shift from original mechanosensing to de novo cell-derived matrix sensing in more physiologically relevant three-dimensional culture is pointed out. A comprehensive understanding of stem cell responses to this stimulus is essential for designing biomaterials that mimic the physiological environment and advancing stem cell-based clinical applications for tissue engineering.