Background. T-cell-depleted bone marrow transplantation (TDBMT) can prevent graft-versus-host disease (GvHD). However, depleting T cells from allogeneic bone marrow often results in failure of bone marrow engraftment under irradiation conditioning. It is not know whether donor T cells are essential for bone marrow engraftment and whether blocking the CD40/CD154 pathway promotes allogeneic TDBM engraftment under nonmyeloablative and irradiation-free fludarabine phosphate and cyclophosphamide conditioning therapy. Methods. Using fully major histocompatibility complex (MHC)-matched mouse models, we investigated whether donor T cells are essential for bone marrow engraftment under fludarabine phosphate and cyclophosphamide conditioning therapy. We also determined whether the barrier of allogeneic TDBM could be overcome by blocking the CD40/CD154 pathway. Donor chimerism was detected by flow cytometric analysis. Donor-specific tolerance through establishing mixed chimerism was tested in vivo by skin transplantation and in vitro by mixed leukocyte reaction and enzyme-linked immunospot (ELISPOT) assay. Results. Compared with unmodified bone marrow, TDBM resulted in poor engraftment when fully MHC-mismatched donors were used. However, anti-CD154 monoclonal antibody (mAb) treatment significantly enhanced donor TDBM engraftment. TDBM engraftment was also seen in CD154 knockout mice. A stable and high level of multilinage donor chimerism was achieved. Recovery of host CD3 T cells was suppressed, and recovery of donor CD3 T cells was promoted, after TDBMT and anti-CD154 mAb treatment. Donor chimerism was established by TDBMT induced donor-specific tolerance in vivo and in vitro. Conclusion. Donor T cells facilitate bone marrow engraftment under nonmyeloablative and irradiation-free conditioning therapy, and the blocking the CD40/CD154 pathway can replace donor T cells to promote TDBM engraftment.