Specific tolerance to allografts has been achieved by a variety of means. We have previously shown that ex vivo removal of dividing CD4+ T cells from an MLR or “pruning” delays skin allograft rejection. We tested pruning of alloreactive T cells as a strategy for retaining a broad T cell repertoire while removing alloreactive T cells in a model of cardiac allograft transplant. Using CFSE staining of responder BALB/c cells with stimulator C57BL/6 cells in an MLR, SCID mice were reconstituted with either dividing (D) or nondividing (ND) CD4+ T cells derived from an MLR and then challenged with heterotopic cardiac allografts. Mice reconstituted with D CD4+ T cells rejected cardiac allografts from the stimulator strain with a median survival time (MST) of 29 days, while mice reconstituted with ND CD4+ T cells maintained allografts from the stimulator strain (MST of> 100 days) while rejecting third-party allografts (B10. BR)(MST= 11 days). ELISPOT assays demonstrate donor-specific hyporesponsiveness of the ND CD4+ T cells. TCR β-chain V region (TRBV) repertoire analysis demonstrates clonal expansion within both rejecting D cardiac allografts and ND cardiac allografts surviving for the long-term. Histology showed greater allograft infiltration by the D CD4+ T cells. The surviving ND cardiac allografts demonstrated reduced cellular infiltration and reduced incidence of allograft vasculopathy, but with the development of chronic fibrosis. Thus, pruning of alloreactive T cells allows long-term-specific cardiac allograft survival while retaining the ability to reject third-party allografts.