Poor survival of human pluripotent stem cells (hPSCs) following freezing, thawing, or passaging hinders maintenance and differentiation in stem cell research. Rho-associated kinases (ROCKs) play a crucial role in hPSC survival. To date, a typical ROCK inhibitor, Y-27632, has been the primary agent used in hPSC research. Here, we report that another ROCK inhibitor, fasudil, can be used as an alternative. Fasudil increased hPSC growth due to survival rather than proliferation following thawing and passaging, similar to Y-27632. It did not affect pluripotency and genetic integrity including mitochondrial genome (mtDNA). Notably, the genes related to metabolism, mTORC1, and TP53 have mainly displayed a faster recovery pattern with ROCK inhibitors than control. Furthermore, fasudil was confirmed as useful for the single dissociation of hPSCs and for aggregation. It also increased retinal pigment epithelium (RPE) differentiation and the survival of neural crest cells during differentiation. These findings suggest that fasudil can replace Y-27632 for use in stem cell research.