Although recent studies have focused on CD4+ regulatory T cells (Treg), CD8+ Treg have also been reported to play important roles in the induction and maintenance of immune tolerance. Adoptive transfer of CD8+ Treg in rodents or induction of CD8+ Treg in humans can prevent or treat allograft rejection and autoimmune diseases. However, no approaches have been reported for the generation of human Ag-specific CD8+ Treg at a practical scale for clinical use. Here, we found that two novel CD8+ T cell subsets with different levels of CD8 surface expression, CD8 high and CD8 low, could be induced from naive CD8+ precursors in vitro by allogeneic CD40-activated B cells, whereas only CD8 high T cells were alloantigen-specific Treg with relatively poor alloantigen-specific cytotoxicity. Importantly, alloantigen-specific CD8 high Treg could be induced and expanded from naive CD8+ CD25− T cells at a large scale after 3 wk of culture without exogenous cytokines. These induced alloantigen-specific Treg were CD45RO+ and CCR7− memory cells, and they expressed Foxp3, CD25, CD27, CD28, and CD62L. The induction and expansion of CD8 high Treg by CD40-activated B cells were dependent on endogenously expressed IFN-γ, IL-2, IL-4, and CTLA-4. This approach may facilitate the clinical application of CD8+ Treg-based immunotherapy in transplantation and autoimmune diseases.