Adoptive transfer of CMV-specific cytotoxic T cells (CTLs) expanded in vitro from memory donor T cells can reduce the incidence of CMV disease in allogeneic transplant recipients. However, this approach has been unavailable in the cord blood (CB) transplantation setting because CB T cells are antigen naive and biased toward Th2/Tc2 function. We developed a protocol to in vitro prime and expand CMV-specific CTLs from CB. T cells were primed with cytokines to trigger skewing toward Th1/Tc1 lineage before encountering monocyte and CD34⁺ progenitor-derived dendritic cells loaded with CMV antigen and its immune complex. CMV-pulsed cultures expanded significantly more over 4 to 6 weeks than CMV cultures despite identical cytokine milieu. T cells isolated from CMV⁺ cultures showed a preferential expansion of CD45RA^-/RO⁺/CD27⁺ T cells compared to CMV^- cultures. CMV-specific IFN-γ- and TNF-α-producing CD4⁺ (Th1) and CD8⁺ (Tc1) T cells were enriched after 3 to 4 weeks and CMV-specific cytotoxicity developed 1 to 2 weeks later.