Despite a rapidly accumulating clinical experience with autologous stem cell transplantation (ASCT) as a treatment for severe refractory autoimmune disease, data on the mechanisms by which ASCT induces immune tolerance are still very scarce. In this study it is shown that ASCT restores immunologic self-tolerance in juvenile idiopathic arthritis (JIA) via 2 mechanisms. First, ASCT induces a restoration of the frequency of FoxP3 expressing CD4⁺CD25^bright regulatory T cells (Tregs) from severely reduced numbers before ASCT to normal levels after ASCT. This recovery is due to a preferential homeostatic expansion of CD4⁺CD25⁺ Tregs during the lymphopenic phase of immunereconstitution, as measured by Ki67 and CD44 expression, and to a renewed thymopoiesis of naive mRNA FoxP3 expressing CD4⁺CD25⁺ Tregs after ASCT. Second, using artificial antigen-presenting cells to specifically isolate self-reactive T cells, we demonstrate that ASCT induces autoimmune cells to deviate from a proinflammatory phenotype (mRNA interferon-γ [IFN-γ] and T-bet high) to a tolerant phenotype (mRNA interleukin-10 [IL-10] and GATA-3 high). These data are the first to demonstrate the qualitative immunologic changes that are responsible for the induction of immune tolerance by ASCT for JIA: the restoration of the CD4⁺CD25⁺ immune regulatory network and reprogramming of autoreactive T cells.