The homeostasis of human pluripotent stem cells (hPSCs) requires the signaling balance of extracellular factors. Exogenous regulators from cell culture medium have been widely reported, but little attention has been paid to the autocrine factor from hPSCs themselves. In this report, we demonstrate that extracellular signal-related kinase 5 (ERK5) regulates endogenous autocrine factors essential for pluripotency and differentiation. ERK5 inhibition leads to erroneous cell fate specification in all lineages even under lineage-specific induction. hPSCs can self-renew under ERK5 inhibition in the presence of fibroblast growth factor 2 (FGF2) and transforming growth factor b (TGF-b), although NANOG expression is partially suppressed. Further analysis demonstrates that ERK5 promotes the expression of autocrine factors such asNODAL, FGF8, andWNT3. The addition of NODAL protein rescuesNANOGexpression and differentiation phenotypes under ERK5 inhibition. We demonstrate that constitutively active ERK5 pathway allows self-renewal even without essential growth factors FGF2 and TGF-b. This study highlights the essential contribution of autocrine pathways to proper maintenance and differentiation.