Dendritic cells (DC) play a key role in the initiation of primary immune response, and pilot clinical studies have demonstrated their ability to induce efficient antitumor immunity. However, the DC used in these clinical trials were generated with various serum sources and were poorly characterized. Obtaining fully characterized DC in controlled and reproducible culture conditions is thus of major interest. We demonstrate that X-VIVO 15 medium supplemented with 2% human albumin can be used to obtain DC. The phenotypic and functional characteristics of these clinical-grade DC were analyzed according to their differentiation stages. CD83 immature DC, obtained in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4, were able to endocyte soluble antigens and internalize apoptotic tumor cells, and also expressed receptors for inflammatory chemokines. Tumor necrosis factor-alpha (TNF-alpha) induced irreversible DC maturation in association with a decreased ability to uptake antigens and an increased allostimulatory capacity. CD83+ mature DC became responsive to EBI1 ligand chemokine (ELC), a chemokine specifically expressed in secondary lymphoid organs. In addition, mature DC obtained with TNF-alpha produced IL-12 and some IL-10 in response to CD40 stimulation. In conclusion, we present well-defined culture conditions allowing the control of DC maturation for clinical or fundamental studies.