Selective depletion of alloreactive T cells from stem-cell allografts should abrogate graft-versus-host disease while preserving beneficial T cell specificities to facilitate engraftment and immune reconstitution. We therefore explored a refined immunomagnetic separation strategy to effectively deplete alloreactive donor lymphocytes expressing the activation antigen CD69 upon stimulation, and examined the retainment of antiviral, antileukemic, and immunoregulatory T cells. In addition to the CD69 high T cell fraction, our studies retrieved two T cell subsets based on residual CD69 expression. Whereas, truly CD69 neg cells were devoid of detectable alloresponses to original stimulators, CD69-low (CD69 low)-expressing T cells elicited significant residual alloreactivity upon restimulation. In interferon-γ enzyme linked immunospot assays, anti-cytomegalovirus and anti-Epstein–Barr virus responses were preserved at significant numbers among CD69 neg T lymphocytes. Accordingly, T cells recognizing the leukemia-associated Wilm's tumor-1 antigen were still detectable in the CD69 neg subset. However, antiviral and antileukemic specificities were also consistently found within CD69 low T cells, suggesting that memory-type donor T cells were partially captured due to residual CD69 expression. Finally, CD4+ CD25+ Foxp3+ immunoregulatory T cells did not upregulate CD69 upon allogeneic stimulation. Our data suggest that CD69-mediated removal of alloreactivity can result in efficient allodepletion, but may partially affect the persistence of antiviral and antileukemic donor memory specificities captured among CD69 low-expressing lymphocytes.