The generation of donor-specific alloantibodies by B cells is detrimental to allograft survival. Recently, a subpopulation of B cells identified as regulatory B cells (Bregs) has been discovered to negatively regulate immune responses. IL-10 cytokine secretion is criticalto Breg function in preventing autoimmune diseases and promoting allograft tolerance. An in-vivo assay for facilitation of hematopoietic stem cell (HSC) engraftment was used to determine the effect of Bregs in facilitating allogeneic HSC engraftment. BALB/c (H2d) mice received 3,000 HSC sorted and 50,000, 25,000, or 12,500 Bregs (CD19+CD1dhiCD5+) from B6 (H2b) mice after ablative total body radiation (950 cGy). Intracellular IL-10 staining was performed on the Breg cells and 10% of these cells were positive for IL-10. We performed a dose-titration of Bregs in in-vivo assay for facilitation. Forty seven percent of recipients of HSC alone engrafted, while groups who received 50,000, 25,000, or 12,500 Bregs plus HSC exhibited superior engraftment with 84.2%, 76.2%, and 68.8%, respectively. However, the addition of 50,000 CD19+CD1d-CD5- non-Bregs resulted in 41.7% donor engraftment which was similar to the engraftment in recipients transplanted with HSC alone. Donor HSC chimerism was multilineage including T cells, B cells, NK cells, dendritic cells, macrophages, and neutrophils. Donor-specifi c tolerance was tested in vitro using one-way MLR. Chimeric T cells were not reactive to donor alloantigens but showed strong proliferation to B10.BR (H2d) MHC-disparate third-party alloantigens. The levels of Vβ5.1/2 and Vβ11expressed by donor B6 CD8+ cells in chimeras were significantly decreased (P < 0.0005) in comparison with B6 naïve mice, suggesting that central deletion of alloreactive T cells is one mechanism for tolerance induction in this model. In conclusion, our data demonstrate that Bregs promote allogeneic HSC engraftment with donor multilineage generation and induce donor-specific transplantation tolerance. DISCLOSURE:Ildstad, S.: Other, Regenerex, LLC, a start-up biotech company, CEO.