We previously showed that one of the amelogenin splicing isoforms, Leucine-rich amelogenin peptide (LRAP), induced osteogenic differentiation of mouse embryonic stein cells; however, the signaling pathway(s) activated by LRAP remained unknown. Here, we demonstrated that the canonical Wnt/beta-catenin signaling is activated upon LRAP treatment, as evidenced by elevated beta-catenin level and increased Writ reporter gene activity. Furthermore, a specific Writ inhibitor sFRP-1 completely blocks the LRAP-mediated Writ signaling. However, exogenous recombinant Wnt3a alone was less effective at osteogenic induction of mouse ES cells in comparison to LRAP. Using a quantitative real-time PCR array, we discovered that LRAP treatment up-regulated the expression of Writ agonists and down-regulated the expression of Wnt antagonists. We conclude that LRAP activates the canonical Writ signaling pathway to induce osteogenic differentiation of mouse ES cells through the concerted regulation of Writ agonists and antagonists. (C) 2009 Elsevier Inc. All rights reserved.