Recombinant poxviruses expressing immunomodulatory molecules together with specific antigens represent powerful vaccines for cancer immunotherapy. Recently, we and others have demonstrated, in vitro and in vivo, that coexpression of CD80 and CD86 costimulatory molecules enhances the immunogenic capacity of a recombinant vaccinia virus (rVV) encoding different tumor-associated antigens. To further investigate the capacity of these vectors to provide ligands for different costimulatory pathways relevant in the generation of T cell responses, we constructed a recombinant virus (rVV) expressing CD40 ligand or CD154 (CD154rVV). Upon binding the CD40 receptor expressed on antigen presenting cells (APC), this molecule, physiologically expressed on activated CD4+ T cells, increases their antigen presentation and immunostimulatory capacities. Therefore, we evaluated the effects of CD154rVV infection on APC activation and its consequences on T cell stimulation. CD154rVV infection of autologous fibroblasts, monocytes, or iDC promoted the expression of a number of cytokines, including GM-CSF, TNF-alpha, and IL-15 in iDC. Most importantly, IL-12 p40 gene expression and protein secretion were induced by CD154rVV but not by wild-type VV (WT VV) in either CD14+ cells or iDC, and these effects could be blocked by anti-CD40 monoclonal antibodies. Furthermore, phenotypic characterization of CD154rVV infected iDC revealed enhanced expression of CD83 and CD86 surface markers as compared with wild-type vaccinia virus infection. As expected, VV infection triggered cytokines gene expression in cultures including APC and T cells from VV immune donors. However, cytokine genes typically expressed by T cell receptor triggered T cells such as those encoding IL-2 and IFN-gamma, or T cell proliferation, were detectable to a significantly higher extent in CD154rVV infected cultures, as compared with WT VV. Activation of specific CD8+ T cells was then investigated using MART-1/Melan-A(27-35) epitope as the model of tumor-associated antigen (TAA). In the presence of CD154rVV activated APCs, significantly higher numbers of specific cytotoxic CD8+ T cells were detected, as compared with cultures performed in the presence of WT VV or in the absence of virus. Taken together, these data indicate that functional CD154 expression from rVV infected cells promotes APC activation, thereby enhancing antigen-specific T cell generation. Such a recombinant vector might help bypass the requirement for activated helper cells during CTL priming, thus qualifying as a potentially relevant vector in the generation of CD8+ T cell responses in cancer immunotherapy.