Pancreatic endocrine β-cells derived from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have received attention as screening systems for therapeutic drugs and as the basis for cell-based therapies. Here, we used a 12-day β-cell differentiation protocol for mouse ES cells and obtained several hit compounds that promoted β-cell differentiation. One of these compounds, mycophenolic acid (MPA), effectively promoted ES cell differentiation with a concomitant reduction of neuronal cells. The existence of neural cell-derived inhibitory humoral factors for β-cell differentiation was suggested using a co-culture system. Based on gene array analysis, we focused on the Wnt/β-catenin pathway and showed that the Wnt pathway inhibitor reversed MPA-induced β-cell differentiation. Wnt pathway activation promoted β-cell differentiation also in human iPS cells. Our results showed that Wnt signaling activation positively regulates β-cell differentiation, and represent a downstream target of the neural inhibitory factor.