Human invariant Vα24+ natural killer T (NKT) cells display potent antitumor activity upon stimulation. Activation of endogenous Vα24+ NKT cells would be one strategy for the treatment of cancer patients. For example, dendritic cells (DCs) loaded with a glycolipid NKT cell ligand, α-galactosylceramide (αGalCer, KRN7000), are a possible tool for the activation and expansion of functional Vα24+ NKT cells in vivo. In this report, we demonstrate that the levels of expansion and the ability to produce IFN-γ of Vα24+ NKT cells induced by αGalCer-loaded whole PBMCs cultured with IL-2 and GM-CSF (IL-2/GM-CSF-cultured PBMCs) were superior to those of cells induced by monocyte-derived CD11c+ DCs (moDCs) developed with IL-4 and GM-CSF. Interestingly, CD11c+ cells in the IL-2/GM-CSF-cultured PBMCs showed a mature phenotype without further stimulation and exerted potent stimulatory activity on Vα24+ NKT cells to enable them to produce IFN-γ preferentially at an extent equivalent to mature moDCs induced by stimulation with LPS or a cytokine cocktail. Cocultivation with CD11c− cells in the IL-2/GM-CSF-cultured PBMCs induced maturation of moDCs. In particular, CD11c−CD3+ T cells appeared to play important roles in DC maturation. In addition, TNF-α was preferentially produced by CD11c−CD3+ T cells in IL-2/GM-CSF-cultured PBMCs and was involved in the maturation of moDCs. Thus, the maturation of DCs induced by CD11c− T cells through TNF-α production appears to result in the efficient expansion and activation of Vα24+ NKT cells to produce IFN-γ preferentially. © 2005 Wiley-Liss, Inc.