In mouse models of asthma, therapeutic use of allergen-presenting IL-10-differentiated dendritic cells (DCs) can abrogate airway hyperresponsiveness, and reduce other asthma-related responses to near background. Analogous human DCs can suppress human T cell responses in vitro, but the operative mechanisms are poorly defined. We investigated the ability of IL-10-treated human DCs to induce tolerance among autologous T cells of subjects with asthma and the mechanisms by which they do this. CD14(+) monocyte-derived DCs were differentiated in the presence of IL-10 (DC10) ex vivo from 11 donors with asthma and 4 control donors, and characterized for relevant markers. They were pulsed with specific or irrelevant allergen, and cultured with autologous peripheral blood CD4(+) T cells, either alone or together with autologous immunostimulatory DCs (DC-TNF), and the impact of this treatment on the T-cell responses was assessed for each donor. The DC10 expressed reduced levels of some relevant markers (CD40, CD80, human leukocyte antigen-DR) and stimulatory cytokines (IL-6 and IL-12), but augmented levels of Ig-like transcript-22/CD85j and IL-10 relative to DC-TNF. In cocultures, they dampened DC-TNF-driven T helper (Th) type 2 cell proliferation and cytokine (IL-4, -5, and -13) secretion. They also drove the development from atopic CD4(+)CD25(lo)Foxp3(lo) cells of a population of IL-10-secreting CD25(+)Foxp3(+)LAG-3(+)CTLA-4(+) regulatory T cells (Tregs). These Tregs suppressed stimulatory DC-induced autologous Th2 cell proliferation and cytokine secretion in a contact-dependent manner. Our data indicate that IL-10-treated human DCs induce Th2 cell allergen tolerance ex vivo by driving the differentiation of Tregs.