It is well established that leukotrienes (LTs), products of the 5-lipoxygenase (5-LO) pathway, participate in inflammatory tissue reactions and immune responses. In the present study, the impact of the 5-LO pathway on vasculogenesis of mouse embryonic stem (ES) cells was investigated.Immunohistochemistry studies demonstrated that 5-LO(+) cells first appeared at day 6 of embryoid body (EB) formation from ES cells. 5-LO(+)/CD68(+) as well as 5-LO(+)/CD45(+) cells were prominent at day 10 of EB differentiation. Real-time PCR and western blot analysis revealed all constituents of the 5-LO pathway. High performance liquid chromatography analyses indicated the synthesis of LTB(4) and LTD(4) in conformity with induction of the 5-LO pathway. Furthermore, Flk-1(+)/CD105(+) cells displayed calcium- (Ca(2+)) transients in response to LTB(4), whereas CD11b(+) cells responded to LTD(4). Treatment of EBs with LTB(4) and LTD(4) resulted in phosphorylation of the mitogen-activated protein kinase ERK1/2. Pharmacological inhibition of the 5-LO pathway and stable shRNA targeting of 5-LO-activating protein decreased capillary cell areas positive for PECAM-1.Our data demonstrate that the 5-LO pathway emerges early during ES cell differentiation into cells of the myeloid lineage and that LTs play an until now unrecognized role in vascular development of ES cells.