The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (Flt3L) is a growth factor for hematopoietic progenitors and induces expansion of the two distinct lineages of dendritic cells (DC) that have been described in humans. These two lineages, DC1 and DC2, have been described according to their ability to induce naive T cell differentiation to T helper cell type 1 (Th1) and Th2 effector cells, respectively. The immunoregulatory potential of DC1 and DC2 depends on their state of maturation and activation, which can be mediated by several molecules. Because monocyte-derived DC1 produce interleukin-12 (IL-12) when stimulated with CD40 ligand (CD40L), we hypothesized that similar results would be obtained with DC1 mobilized by Flt3L. Unexpectedly, we found that immature DC expanded in vivo by Flt3L treatment could not be stimulated to produce IL-12 in vitro using CD40L and/or interferon-gamma (IFN-gamma) alone. Instead, we found that Flt3L-mobilized DC from cancer patients require a sequence of specific signals for maturation, which included initial treatment with granulocyte macrophage-colony stimulating factor followed by a combination of maturation signals such as CD40L and IFN-gamma. Flt3L-mobilized DC matured in this manner possessed greater T cell-stimulatory function than nonmatured DC. The ability to generate phenotypically mature, IL-12-producing DC1 from peripheral blood mononuclear cells mobilized by Flt3L will have important implications for the development of effective cancer immunotherapy strategies.