Donor-reactive memory T cells present a special hurdle in transplantation. Although hematopoietic chimerism is effective for inducing donor-specific tolerance, the effects on memory T cells are unclear. Here, we induced stable chimerism and tolerance in mice (Tolerance group, n = 6) by donor-specific transfusion (DST) plus anti-CD154 monoclonal antibody (mAb), avoiding the toxic myeloablative conditioning treatment to assist bone marrow transplantation (DST/aCD154&BMTx). We then transferred memory CD4(+) or CD8(+) T cells from donor antigen primed mice to the tolerance-induced recipients 4 days after heart transplantation (Tol/CD4(+) Tm group and Tol/CD8(+) Tm group, n = 6, respectively), but neither of these memory T-cell subsets had an effect on the permanent graft survival (median survival time > 100 days). The unaltered rate of memory T cells in spleen and anergy to donor antigen in vitro demonstrated that these memory T cells were well controlled. The chimerism-promoting protocol DST/aCD154&BMTx produced an immune environment that included high levels of regulatory T cells (Tregs), microchimerism and TGF-beta, all of which may act in suppressing the donor-reactive memory CD4(+) or CD8(+) T cells. These findings have potentially important implications for designing approaches to suppressing memory T cells for success of transplantation.