Recently it has been shown that dendritic cells (DC) can develop from peripheral blood monocytes when grown in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). However, it is unclear whether DC can also develop from monocytes in absence of these cytokines. We therefore analyzed the effect of Flt-3 ligand (Flt3L) and of CD40 ligand on the development of human DC from blood monocytes in the absence of GM-CSF. Adherent peripheral blood mononuclear cells (PBMNC) were cultured in the presence of different cytokine combinations and analyzed for the expression of surface molecules and antigen presenting capacity. For functional analyses, cells were tested for their ability to stimulate allogeneic T lymphocytes in a mixed lymphocyte reaction (MLR), to present soluble antigens, and to induce primary HIV-peptide–specific cytotoxic T-cell (CTL) responses in vitro. Furthermore, expression of DC-CK1, a recently identified chemokine with specific expression in DC, and of IL-18 (IGIF), a growth and differentiation factor for Th 1 lymphocytes, was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). In our study, Flt3L alone was not sufficient to generate DC and required addition of IL-4. DC generated with Flt3L and IL-4 underwent maturation after stimulation with tumor necrosis factor- (TNF-) or CD40L, characterized by CD83 expression, upregulation of MHC, adhesion, and costimulatory molecules as well as increased allogeneic proliferative response. In contrast, CD40 ligation alone promoted differentiation of adherent blood monocytes into functional DC in the absence of GM-CSF and IL-4. These cells displayed all phenotypic and functional characteristics of mature DC and were potent stimulatory cells in priming of major histocompatibility complex (MHC) class I–restricted CTL responses against an HIV-peptide, whereas their ability to present soluble protein antigens was reduced. Using a semiquantitative RT-PCR, DC-CK1 and IL-18 transcripts were detected in all generated DC populations, independent of growth factors used. Our findings provide further evidence for the importance of CD40-CD40L interaction for initiation and maintenance of T-cell responses and confirm the emerging concept that blood monocytes provide an additional source of DC depending on external stimuli.