Dendritic cells (DC), potent antigen presenting cells capable of activating both naïve and primed T cells, are currently being pursued clinically in the development of cancer vaccines. Variations in the literature regarding DC source, culture conditions, maturation state, dose, and route of immunization make comparisons of clinical trial data difficult. In order to define and optimize the culture conditions for DC generation, we have performed a careful comparison of two culture methods, as well as different methods of DC maturation. Our studies demonstrate that high viability DC can be produced and matured in gas permeable hydrophobic culture bags. These cells express surface molecules characteristic of DC and have superior yield, viability, and function to cells cultured in plastic tissue culture flasks. These results suggest that hydrophobic culture bags are ideal for the preparation of clinical DC vaccines, as DC can be generated, antigen-loaded, and matured in a closed system, a scheme we have found to be superior to previously described flask culture methods.