Immunotherapeutic approaches to limit cytomegalovirus (CMV) morbidity and mortality after hematopoietic stem cell transplants (HSCTs) are currently under investigation as alternatives to antiviral drugs. In this context, we have inserted full-length and ubiquitin-modified CMV phosphoprotein 65 (pp65), phosphoprotein 150 (pp150), and immediate early protein 1 (IE1) immunodominant antigens into the virulent Western Reserve strain of vaccinia virus (VV) and the highly attenuated strain, modified vaccinia Ankara (MVA). Recombinant (r) VV or rMVA stimulated vigorous expansion of CMV-specific CD8+ T cells in CMV-positive donor peripheral blood mononuclear cells (PBMCs), which showed minimal alloreactivity and high levels of HLA tetramer binding, cytokine production, and cytotoxicity. Ubiquitinated antigens had a profound effect when expressed in VV. Single antigen rMVA expressing pp65 or IE1, either ubiquitin-modified or native, stimulated both cytotoxic T lymphocyte (CTL) populations to be expanded up to 500-fold in a 60-mL blood draw from the same donor. This result demonstrates the clinical feasibility of simultaneously amplifying multiple CMV-CTL populations. Transgenic HLA A2.1 (HHD II) mice, immunized with the same rMVA as used with human PBMCs, produced a robust cytotoxic response to both CMV pp65 and IE1. The specificity of the vigorous immunologic response to rMVA, both in vitro and in vivo, makes them candidates for clinical evaluation in the context of adoptive immunotherapy for hematopoietic stem cell transplant (HSCT) recipients or donor vaccination.