SmartDCs (Self-differentiated Myeloid-derived Antigen-presenting-cells Reactive against Tumors) consist of highly viable dendritic cells (DCs) induced to differentiate with lentiviral vectors (LVs) after an overnight ex vivo transduction. Tricistronic vectors co-expressing cytokines (granulocyte-macrophage-colony stimulating factor [GM-CSF], interleukin [IL]-4) and a melanoma antigen (tyrosine related protein 2 [TRP2]) were used to transduce mouse bone marrow cells or human monocytes. Sixteen hours after transduction, the cells were dispensed in aliquots and cryopreserved for identity, potency, and safety analyses. Thawed SmartDCs readily differentiated into highly viable cells with a DC immunophenotype. Prime/boost subcutaneous administration of 1×10(6) thawed murine SmartDCs into C57BL/6 mice resulted into TRP2-specific CD8(+) T-cell responses and protection against lethal melanoma challenge. Human SmartDC-TRP2 generated with monocytes obtained from melanoma patients secreted endogenous cytokines associated with DC activation and stimulated TRP2-specific autologous T-cell expansion in vitro. Thawed human SmartDCs injected subcutaneously in NOD.Rag1(-/-).IL2rγ(-/-) mice maintained DC characteristics and viability for 1 month in vivo and did not cause any signs of pathology. For development of good manufacturing practices, CD14(+) monocytes selected by magnetic-activated cell separation were transduced in a closed bag system (multiplicity of infection of 5), washed, and cryopreserved. Fifty percent of the monocytes used for transduction were recovered for cryopreservation. Thawed SmartDCs produced in two independent runs expressed the endogenous cytokines GM-CSF and IL-4, and the resulting homogeneous SmartDCs that self-differentiated in vitro contained approximately 1.5-3.0 copies of integrated LVs per cell. Thus, this method facilitates logistics, standardization, and high recovery for the generation of viable genetically reprogrammed DCs for clinical applications.