Human mast cells (hMCs) derived in vitro from cord blood mononuclear cells exhibit stem cell factor (SCF)-dependent comitogenic responses to T helper cell type 2 (Th2) cytokines. As cysteinyl leukotriene (cys-LT) biosynthesis is a characteristic of immunoglobulin (Ig)E-activated mucosal hMCs, we speculated that Th2 cytokines might regulate eicosanoid generation by hMCs. After passive sensitization for 5 d with IgE in the presence of SCF, anti-IgE–stimulated hMCs elaborated minimal cys-LT (0.1 ± 0.1 ng/10⁶ hMCs) and abundant prostaglandin (PG)D₂ (16.2 ± 10.3 ng/10⁶ hMCs). Priming of hMCs by interleukin (IL)-4 with SCF during passive sensitization enhanced their anti-IgE–dependent histamine exocytosis and increased their generation of both cys-LT (by 27-fold) and PGD₂ (by 2.5-fold). Although priming with IL-3 or IL-5 alone for 5 d with SCF minimally enhanced anti-IgE–mediated cys-LT generation, these cytokines induced further six- and fourfold increases, respectively, in IgE-dependent cys-LT generation when provided with IL-4 and SCF; this occurred without changes in PGD₂ generation or histamine exocytosis relative to hMCs primed with IL-4 alone. None of these cytokines, either alone or in combination, substantially altered the levels of cytosolic phospholipase A₂ (cPLA₂), 5-lipoxygenase (5-LO), or 5-LO activating protein (FLAP) protein expression by hMCs. In contrast, IL-4 priming dramatically induced the steady-state expression of leukotriene C₄ synthase (LTC₄S) mRNA within 6 h, and increased the expression of LTC₄S protein and functional activity in a dose- and time-dependent manner, with plateaus at 10 ng/ml and 5 d, respectively. Priming by either IL-3 or IL-5, with or without IL-4, supported the localization of 5-LO to the nucleus of hMCs. Thus, different Th2-derived cytokines target distinct steps in the 5-LO/LTC₄S biosynthetic pathway (induction of LTC₄S expression and nuclear import of 5-LO, respectively), each of which is necessary for a full integrated functional response to IgE-dependent activation, thus modulating the effector phenotype of mature hMCs.