Lentiviral vectors are promising vaccine vector candidates that have been tested extensively in preclinical models of infectious disease and cancer immunotherapy. They are also used in gene therapy clinical trials both for the ex vivo modification of cells and for direct in vivo injection. It is therefore critical to understand the mechanism(s) by which such vectors might stimulate the immune system. We evaluated the effect of lentiviral vectors on myeloid dendritic cells (DC), the main target of lentiviral transduction following subcutaneous immunization. The activation of DC cultures was independent of the lentiviral pseudotype but dependent on cell entry and reverse transcription. In vivo-transduced DC also displayed a mature phenotype, produced tumor necrosis factor alpha (TNF-alpha), and stimulated naive CD8(+) T cells. The lentiviral activation of DC was Toll-like receptor (TLR) dependent, as it was inhibited in TRIF/MyD88 knockout (TRIF/MyD88(-/-)) DC. TLR3(-/-) or TLR7(-/-) DC were less activated, and reverse transcription was important for the activation of TLR7(-/-) DC. Moreover, lentivirally transduced DC lacking TLR3 or TLR7 had an impaired capacity to induce antigen-specific CD8(+) T-cell responses. In conclusion, we demonstrated TLR-dependent DC activation by lentiviral vectors, explaining their immunogenicity. These data allow the rational development of strategies to manipulate the host's immune response to the transgene.