The tumor milieu can influence dendritic cell (DC) differentiation. We analyzed DC differentiation in a 3-dimensional tumor model and propose a new mechanism of DC modulation by the tumor environment. Monocytes were cultured in the presence of IL-4 and GM-CSF within multicellular tumor spheroids (MCTSs) generated from different tumor cell lines. Monocytes invaded the MCTSs and differentiated into tumor-associated dendritic cells (TADCs). The antigen expression was altered on TADCs independent of the culture conditions (immature/mature DCs, Langerhans cells) and IL-12 secretion was reduced. Supernatants of MCTSs could partially transfer the suppressive effect. Conditioned media from urothelial carcinoma cell lines contained high levels of M-CSF and IL-6, both cytokines known to modulate DC differentiation. In contrast, melanoma and prostate carcinoma MCTS cocultures produced little M-CSF and IL-6, but high levels of lactic acid. Indeed, addition of lactic acid during DC differentiation in vitro induced a phenotype comparable with TADCs generated within melanoma and prostate carcinoma MCTSs. Blocking of lactic acid production in melanoma MCTS cocultures reverted the TADC phenotype to normal. We therefore conclude that tumor-derived lactic acid is an important factor modulating the DC phenotype in the tumor environment, which may critically contribute to tumor escape mechanisms.