Culture conditions for human dendritic cells (DC) have been developed by several laboratories. Most of these culture methods, however, have used conditions involving fetal calf serum (FCS) to generate DC in the presence of granulocyte‐macrophage colony‐stimulating factor and interleukin (IL)‐4. Recently, alternative culture conditions have been described using an additional stimulation with monocyte‐conditioned medium (MCM) and FCS‐free media to generate DC. As MCM is a rather undefined cocktail, the yield and quality of DC generated by these cultures varies substantially. We report that a defined cocktail of tumor necrosis factor (TNF)‐α, IL‐1β and IL‐6 equals MCM in its potency to generate DC. Addition of prostaglandin (PG)E₂ to the cytokine cocktail further enhanced the yield, maturation, migratory and immunostimulatory capacity of the DC generated. More importantly, culture conditions also influenced the outcome of the T cell response induced. DC cultured with TNF‐α/IL‐1/IL‐6 or MCM alone induced CD4⁺ T cells that release intermediate levels of interferon (IFN)‐γ and no IL‐4 or IL‐10. Production of IFN‐γ was significantly induced by addition of PGE₂, while no effect on production of IL‐4 or IL‐10 was observed. Even more striking differences were observed for CD8⁺ T cells. While MCM conditions only induced IFN‐γ^low, IL‐4^neg cells, TNF‐α/IL‐1/IL‐6 promoted growth of IFN‐γ^intermediate, IL‐4^neg CD8⁺ T cells. Addition of PGE₂ again only further polarized this pattern enhancing IFN‐γ production by alloreactive CD8⁺ T cells in both cultures without inducing type 2 cytokines. Taken together, the data indicate that the defined cocktail TNF‐α/IL‐1/IL‐6 can substitute for MCM and that addition of PGE₂ further enhances the yield and quality of DC generated. TNF‐α/IL‐1, IL‐6 + PGE₂‐cultured DC seem to be optimal for generation of IFN‐γ‐producing CD4/CD8⁺ T cells.