The major complications of allogeneic blood and marrow transplantation (BMT) include graft failure, opportunistic infections, leukemic relapse and graft-versus-host disease (GVHD). The incidence of the first three complications is diminished by the presence of mature donor T cells in the transplant inoculum. Unfortunately, the transplantation of mature alloreactive donor T cells also directly induces the latter complication of acute GVHD, which can be directed against either HLA or minor histocompatibilty antigen (miHA) disparities. Efforts to deplete donor marrow inoculum of T cells have been successful in minimizing the development of disease but have been associated with increased leukemic relapse. Thus, one approach to solve this problem would be to identify and then selectively deplete alloreactive donorT cells while allowing the residual T cells to provide protection against infection and to mediate a leukemia-specific GVL response. In this regard, TCR Vβ repertoire analysis by CDR3-size spectratyping can be a powerful tool to characterize the alloreactive T cell response. As an initial test of this capability, we studied lethal GVHD in the B6->CXB-2 MHC-matched murine model, expressing multiple miHA differences. Herein, we determined the correlation between the in vitro alloreactive-specific CD8+ T cell response and their actual involvement in the development of GVHD. The results indicated a high correlation in the Vβ CDR3-size skewing patterns between the in vitro and in vivo spectratype histograms, and laid the foundation for the potential clinical application of this approach. We proceeded to test the predictive value of the in vitro spectratyping assays of donor anti-host mixed lymphocyte reactions with GVHD outcome in the clinical BMT setting. The results again indicated a high association of overlapping CDR3-size skewing. Thus, in vitro spectratyping analysis may be useful in guiding the manipulation of the donor T cell inoculum in order to provide improved BMT outcomes.