Dendritic cells (DC) are powerful antigen-presenting cells that have drawn many attentions due to the recent development of anti-cancer vaccines. Clinical grade production of monocyte-derived DC (Mo-DC) is extensively studied, and many efforts are made to develop and improve clinical standard operating procedures. Most of the parameters involved, such as the cytokines and maturation agents, have been widely assessed. However, very few are investigated about how culture medium and additional protein components affect DC yield, viability and maturation. Thus, our study aimed to compare the impact of standard culture medium on Mo-DC differentiation and maturation. Commercially available media for hematopoietic cell culture as well as different protein supplementations, that is foetal calf serum (FCS), autologous plasma (AP), human serum (HS) and human serum albumin (HSA) were tested. Culture yields, cell viability and DC maturation were investigated. Differentiation yields were similar between the conditions used. However, we evidenced significant differences in terms of cytotoxicity and DC maturation (phenotypic and functional). This underscores the importance of defining culture medium composition in clinical standard operating procedures to insure quality control, and also when preparing DC for experimental uses.