Previous studies have established that adoptive immunotherapy with donor-derived virus-specific T cells can prevent/treat viral complications post-stem cell transplant and regulatory T cells show promise as inhibitors of graft-versus-host disease. On the basis of flow cytometric analysis of upregulation of activation markers after stimulation with viral peptide pools, we have developed a rapid and clinically applicable protocol for the simultaneous selection of virus-specific T cells (after stimulation with peptide pools for the immunodominant antigens of cytomegalovirus, Epstein-Barr virus, and adenovirus) and regulatory T cells using CD25 immunomagnetic selection. Using tetramer staining, we detected enrichment of CD8 T cells recognizing peptide epitopes from cytomegalovirus and Epstein-Barr virus antigens after CD25 selection in 6 of 7 donors. Enzyme-linked immunospot assays demonstrated the simultaneous presence of bivirus-specific or trivirus-specific cells in all evaluated donors, with a median 29-fold (6 to 168), 40-fold (1 to 247), and 16-fold (1 to 219) enrichment of cells secreting interferon-γ in response to cytomegalovirus pp65, adenovirus hexon, and Epstein-Barr virus lymphoblastoid cells compared with unmanipulated peripheral blood mononuclear cells from the same donors. Furthermore, the CD25-enriched cells lost alloreactivity in H-thymidine proliferation assays and showed highly effective (median, 98%) suppression of alloreactivity in all evaluated donors. In summary, we have developed a rapid, simple Good Manufacturing Practice compliant methodology for the simultaneous selection of T cells with multiple viral specificities and regulatory T cells. Adoptive transfer of T cells generated using this strategy may enable restoration of cellular immunity to viruses after allogeneic stem cell transplant with a low risk of graft-versus-host disease. Owing to the speed and simplicity of this methodology, this approach may significantly broaden the applicability of adoptive immunotherapy.