The actual leukotriene D4 (LTD4) signaling pathways that regulate cell proliferation have not been elucidated thoroughly although fatty acid and its metabolites play a key role in regulations of embryonic functions. Thus, this study investigated the response of mouse embryonic stem (ES) cells exposed to LTD4 and elucidated the signaling pathways as well. LTD4 increased DNA synthesis in concentration-dependent (≥10−7 M) and time-dependent (≥12 h) manners, as determined by [3H] thymidine incorporation and increased cell number. LTD4 induced the phosphorylation of signal transducer and activator of transcription-3 (STAT3) and the increase of intracellular Ca2+ levels via cysteinyl leukotriene (CysLT) 1 and 2 receptors. LTD4 increased Akt activation and calcineurin expression, which were blocked by STAT3 inhibitor and calcium chelators. LTD4-induced glycogen synthase kinase (GSK)-3β phosphorylation was decreased by LY294002, Akt inhibitor, and cyclosporine A. LTD4 inhibited the phosphorylation of β-catenin. In addition, LTD4-stimulated migration through increased activation of focal adhesion kinase (FAK) and paxillin which were blocked by Akt inhibitor and cyclosporine A. LTD4-induced increases in protooncogene and cell cycle regulatory proteins were blocked by cyclosporine A, FAK siRNA, and β-catenin siRNA. In conclusion, LTD4-stimulated mouse ES cell proliferation and migration via STAT3, phosphoinositide 3-kinases (PI3K)/Akt, Ca2+-calcineurin, and GSK-3β/β-catenin pathway. J. Cell. Biochem. 111: 686–698, 2010. © 2010 Wiley-Liss, Inc.