Dendritic cells (DCs) are the most potent antigen presenting cells and are therefore widely used in cancer immunotherapy. An optimal method for the generation of DCs for clinical use remains to be established. The aim of the study was to find a serum-free media (SFM) able to generate reproducible and functional cultures of DCs for clinical studies. We characterized immature and mature DCs cultured in SFM, CellGro DC and X-VIVO15, and serum media (SM), RPMI 1640+5% human serum or autologous serum. The expression of HLA-DR, CD86, CD83 was higher in SM-cultured DCs (SM-DCs) than SFM-derived DCs (SFM-DCs). Between SFM-DCs, CellGro-cultured DCs (CellGro-DCs) showed a higher expression and an improved up-regulation capacity of all molecules as compared with X-VIVO15-derived DCs (X-VIVO15-DCs). CellGro-DCs and SM-DCs showed a similar mannose receptor expression and related endocytic capacity tested by fluorescein isothiocyanate-dextran uptake. In contrast X-VIVO15-DCs expressed low levels of mannose receptor and were unable to endocyte fluorescein isothiocyanate-dextran. DCs cultured in all conditions stimulated a mix lymphocyte reaction, but CellGro-DCs and SM-DCs induced a more potent T-cell proliferation compared with X-VIVO15-DCs. Cytokine analysis showed that after maturation, all DC cultures produced IL-12p70 and IL-10 except for X-VIVO15-DCs which only produced the latter cytokine. SM-DCs and SFM-DCs induced a TH1 polarization in allogeneic naive T cells. In conclusion, a comparative analysis of DC performance generated in different conditions allows us to determine CellGro DC as the optimal medium for the generation of clinical grade DCs.