Abstract:  Dendritic cells (DC) are potent antigen‐presenting cells capable to induce efficient antigen‐specific T cell responses in vitro and in vivo. Herein, the maturation process is of great significance, as immature DC (iDC) are known to induce rather regulatory than effector T cell differentiation. This study was designed to characterize the role of the CD40‐CD40L pathway for differentiation and function of human DC. Therefore, iDC were stimulated through CD40‐CD40L interaction by transduction of DC with adenoviral vectors encoding for CD40L (Ad‐CD40L). Resulting DC (CD40L‐DC) were analysed concerning their phenotype, cytokine profile and T cell stimulatory capacity. Transduction induced a DC phenotype comparable to stimulation with proinflammatory cytokines as revealed by upregulation of CD83 and the costimulatory molecules CD80 and CD86. Additionally, Ad‐CD40L‐induced strong production of IL‐12p70 not observed in cytokine‐matured DC. Surprisingly, the T cell stimulatory capacity was markedly reduced in CD40L‐DC. Furthermore, stimulation of CD8⁺ T cells by peptide‐loaded CD40L‐DC resulted in a substantial reduction of antigen‐specific IFN‐γ production. This phenomenon is due to an enhanced IL‐10 production of CD40L‐DC in DC‐T cell coculture as well as a stabilization of the IL‐10 receptor expression on activated T cells. These data demonstrate that DC stimulated through CD40‐CD40L interaction differentiate into tolerogenic DC with immunomodulatory function.