Non-human primates are suitable models for preclinical research aimed at cell-replacement therapies. Recently, it has been reported that Rho-associated kinase inhibitor Y-27632 markedly reduced dissociation-induced apoptosis of human embryonic stem (hES) cells, and is expected as a novel supplement for hES cell maintenance or differentiation inductions; however, the effects of the chemical are still to be determined in model animals. Here, we demonstrated the effect of Y-27632 on cynomolgus monkey ES (cyES) cells. Also, in cyES cells, Y-27632 treatment dramatically improved the efficiency of colony formation from single cells without affecting the pluripotent state and karyotype. Y-27632 supplementation was also effective for feeder-free culture and differentiation induction. Neural stem cells directly induced from cyES cells could give rise to neurons, astrocytes and dopamine producing cells. The present result not only suggests that the chemical was effective for improving the culture system of primate ES cells, but also the similarity between cyES and hES cells regarding the reactions to the chemical, which might be further evidence that cyES cells are superior models for hES cells.