The clinical usage of dendritic cells (DC) for tumor immunotherapy still requires improvements. In this study, three alternative maturation mixtures were compared with the cytokine-based gold standard, and the overall interaction of the resulting DC with effector cells from the innate as well as the adaptive immunity was evaluated in healthy donors. Stimulation with the TLR-4 ligand monophosphoryl lipid A together with IFN-γ (alt-2 DC) resulted in DC with the highest levels of costimulatory molecule expression and IL-12p70/IL-10 ratio. Whereas all alternative DC were able to induce NK and γδ T cells to acquire cytotoxic properties and secrete type 1 and proinflammatory cytokines, after both short (20-h)- and long (5-8 d)-time coculture, secretion of IFN-γ by the innate populations was induced in response to alt-2 and alt-1 DC (TNF-α, IFN-α, IFN-γ, IL-1β, poly IC), but not to alt-3 DC (TNF-α, IFN-γ, IL-1β, CL097). Regarding CD8(+) T cell-mediated Ag-specific immune responses, a heterogeneous pattern of responses was obtained among the healthy donors, suggesting rather a competition than a synergy among the different effector cells. Our data promote further evaluation of alt-2 fast DC for translatability into clinical immunotherapy trials, while also fostering the need to identify biomarkers for immune cell responsiveness and tumor susceptibility to be able to select for each patient the best possible DC-based therapy.