The ability of dendritic cells (DCs) to trigger tolerance or immunity is dictated by the context in which an antigen is encountered. A large body of evidence indicates that antigen presentation by steady-state DCs induces peripheral tolerance through mechanisms such as the secretion of soluble factors, the clonal deletion of autoreactive T cells, and feedback control of regulatory T cells. Moreover, recent understandings on the function of DC lineages and the advent of murine models of DC depletion have highlighted the contribution of DCs to lymphocyte tolerance. Importantly, these findings are now being applied to human research in the contexts of autoimmune diseases, allergies, and transplant rejection. Indeed, DC-based immunotherapy research has made important progress in the area of human health, particularly in regards to cancer. A better understanding of several DC-related aspects including the features of DC lineages, milieu composition, specific expression of surface molecules, the control of signaling responses, and the identification of competent stimuli able to trigger and sustain a tolerogenic outcome will contribute to the success of DC-based immunotherapy in the area of lymphocyte tolerance. This review will discuss the latest advances in the biology of DC subtypes related to the induction of regulatory T cells, in addition to presenting current ex vivo protocols for tolerogenic DC production. Particular attention will be given to the molecules and signals relevant for achieving an adequate tolerogenic response for the treatment of human pathologies.