Dendritic cells (DCs) play a key role in the orchestration of immune reactions. Manipulation of DC function through genetic manipulation for vaccine development provides a multitude of applications for active immunotherapy of cancer and chronic infections. Several laboratories have shown that lentiviral vectors (LVs) are efficient and consistent tools for ex vivo gene manipulation of DCs and their precursors. LVs integrate in the genome of target cells resulting in persistent and stable transgene expression, and gene delivery does not result in cytostatic or nonspecific adverse immunomodulatory reactions. Mouse, macaque, and human DCs are efficiently transduced with LVs, allowing preclinical vaccination studies to be gradually implemented into clinical trials. This chapter describes HIV-1-derived LV transduction used for ex vivo gene delivery of marking genes, antigens, and immunomodulatory molecules into mouse and human hematopoietic precursors and DCs. With the perspective of bioengineering DCs from the inside-out, we also describe a one-hit LV transduction method for constitutive expression of GM-CSF and IL-4 genes, which allows self-differentiation of mouse and human hematopoietic precursor cells into highly viable and potent DCs.