Recent investigations suggest that hypoxia increases the release of fatty acids, which participate in the regulation of cytokine synthesis and cell growth. Therefore, in this study, we examined the effect of arachidonic acid (AA) on hypoxia-induced vascular endothelial growth factor (VEGF) expression and its related signaling pathways in mouse embryonic stem (ES) cells. Hypoxia increased the level of [(3)H]AA release and VEGF expression. AA treatment concurrent with hypoxia further increased the PGE(2) production and VEGF expression level, which was inhibited by the suppression of cPLA(2) and cyclooxygenase 2 (COX-2) pathways. Hypoxia increased the level of Notch-1 and Wnt-1/beta-catenin expression, which was blocked by the inhibition of COX-2, and inhibition of Notch-1 by gamma-secretase inhibitor blocked Wnt-1 activation. Moreover, the hypoxia-induced increase of hypoxia-inducible factor 1alpha (HIF-1alpha) expression induced Notch-1 activation and was regulated by Wnt-1 activation. The expression of each signaling molecule induced an increase in VEGF expression that was greater in hypoxia with AA than in hypoxia alone. The inhibition of VEGF expression using VEGF-targeted small interfering RNA decreased the hypoxia-induced increase in cell cycle regulatory protein expression, DNA synthesis, and cell number, suggesting that hypoxia-induced VEGF expression stimulates proliferation of mouse ES cells. In conclusion, AA potentiates hypoxia-induced VEGF expression in mouse ES cells through the Notch-1, Wnt-1, and HIF-1alpha pathways.