Integrase-defective lentiviral vectors (ID-LVs) show several hallmarks of conventional lentiviral vectors such as absence of cytotoxic effects and long-term expression in non-replicating target cells. The integration rate of ID-LVs into the genome of target cells is dramatically reduced, which enhances safety and opens perspectives for their use in vaccine development. ID-LVs have been shown to be effective vaccines in mouse models, but functional studies with human cells in vitro and in vivo are lacking. Here, we evaluated whether ID-LVs expressing combinations of cytokines (GM-CSF/IL-4 or GM-CSF/IFN-α) used to transduce human monocytes would result in functional "induced dendritic cells" (iDCs). Overnight transduction of monocytes with high titer ID-LVs generated highly viable (14 days) and immunophenotypically stable iDCs expressing GM-CSF/IL-4 ("SmartDCs") or GM-CSF/IFN-α ("SmyleDCs"). SmartDCs and SmyleDCs maintained in vitro continuously secreted the transgenic cytokines and showed up-regulation of several endogenously produced inflammatory cytokines (IFN-γ, IL-2, -5, -6, and -8). Both iDC types potently stimulated T cells in mixed lymphocyte reactions at levels comparable to conventional DCs (maintained with exogenous cytokines). A single in vitro stimulation of CD8(+) T cells with autologous SmartDCs or SmyleDCs pulsed with peptide pools of pp65 (a human cytomegalovirus antigen) resulted in high expansion of central memory and effector memory CTLs reactive against different pp65 epitopes. We further evaluated the effects of SmartDCs and SmyleDCs to expand anti-pp65 CTLs in vivo using immune deficient NOD/Rag1((-/-))/IL-2rγ((-/-)) (NRG) mice. NRG mice immunized subcutaneously with SmartDCs or SmyleDCs co-expressing the full-length pp65 were subsequently infused with autologous CD8(+) T cells. Both types of iDCs effectively stimulated human CTLs reactive against different pp65 antigenic determinants in vivo. Due to the simplicity of generation, robust viability and combined capacity to stimulate homeostatic, antigenic and multivalent responses, iDCs are promising vaccines to be explored in immunization of lymphopenic patients in the post-transplantation setting.