Rho kinase (ROCK) regulates the functions of several target proteins via its kinase activity. Therefore, ROCK activity inhibition may provide new possibilities of controlling the in vitro neuronal differentiation of embryonic stem (ES) cells. When we investigated the effects of the ROCK inhibitor Y-27632 on ES cell differentiation, we found that this inhibitor promoted the differentiation of these cells into neurons. Furthermore, we found that ROCK inhibition may promote the neuronal differentiation of ES cells by activating extracellular signal-regulated kinase (ERK) involved in the ERK signaling pathway. In this study, we investigated the effects of specific inhibitors of several cellular signaling components on the promotion of neuronal differentiation in ES cells to clarify the roles of cellular signaling pathways in the ROCK inhibitor-mediated cell differentiation process. Our results suggest that ERIC may be activated via the Ras/Raf/MEK, the PI3K/PKC, or the Cdc42/Rac signaling pathways in the ROCK inhibitor-mediated promotion of neuronal differentiation in ES cells. (C) 2016 Elsevier Ireland Ltd. All rights reserved.