Research on human pluripotent stem cells (hPSCs) has expanded rapidly over the last two decades, owing to the promises of hPSCs for applications in regenerative medicine, disease modeling, and developmental biology studies. While most studies of hPSCs have so far focused on identifying extrinsic soluble factors, intracellular signaling pathways, and transcriptional networks that are involved in regulating hPSC self-renewal and differentiation, a few promising studies have emerged in recent years to reveal some unique mechano-sensitive and -responsive properties of hPSCs and the effect of the physical aspects of the local cellular microenvironment on regulating hPSC behaviors. This Frontier Review is to highlight these recent studies of mechanobiology in hPSCs and to discuss the impact of advancing our understanding of mechanoregulation of hPSC behaviors on improving survival, self-renewal and differentiation of hPSCs using well-controlled synthetic micro/nanoscale cell culture tools.