Donor-specific CD4+CD127-CD25+FOXP3+ regulatory T cells (AgTregs) have the potential to induce clinical transplant tolerance; however, their expansion ex vivo remains challenging. We optimized a novel expansion protocol to stimulate donor-specific Tregs using soluble 4-trimer CD40 ligand (CD40L)-activated donor B cells that expressed mature antigen-presenting cell markers. This avoided the use of CD40L-expressing stimulator cells that might otherwise result in potential cellular contamination. Purified allogeneic "recipient" CD4+CD25+ Tregs were stimulated on days 0 and 7 with expanded "donor" B cells in the presence of IL-2, TGFβ and sirolimus (SRL). Tregs were further amplified by polyclonal stimulation with anti-CD3/CD28 beads on day 14 without SRL, and harvested on day 21, with extrapolated fold expansion into the thousands. The expanded AgTregs maintained expression of classical Treg markers including demethylation of the Treg-specific demethylated region (CNS2) and also displayed constricted TcR repertoire. We observed AgTregs more potently inhibited MLR than polyclonally expanded Tregs and generated new Tregs in autologous responder cells (a measure of infectious tolerance). Thus, an optimized and more clinically applicable protocol for the expansion of donor-specific Tregs has been developed.