Objective. Recent reports have shown that donor or host CD4 +CD25+ Treg cells can be used to control GVHD or graft rejection following allogeneic BMT in mice. In the present study we investigated the potential of third-party Treg cells compared to donor-type cells to facilitate BM allografting. Methods. Graft rejection is assessed in a mouse model of T cell-mediated BM allograft rejection. Lethally irradiated C3H mice are transplanted at day 2 after irradiation with T cell-depleted Balb/Nude BM. Graft rejection is induced by purified host-type T cells infused one day prior to BMT. Cells tested for their facilitating activity are added to the T cell-depleted BM allograft. Results. Naïve or ex vivo-expanded third-party Treg cells can effectively enhance engraftment of T cell-depleted BM allografts, exhibiting reactivity in vitro and in vivo similar to that found for donor-type Treg cells. Conclusion. The use of third-party Treg cells in contrast to donor-type cells could allow advanced preparation of a large bank of Treg cells (off-the-shelf), with all the appropriate quality controls required for cell therapy.