Fms-like tyrosine kinase 3 ligand (Flt3L) is a growth factor for dendritic cells (DCs), hematopoietic stem cells, and natural killer (NK) cells in vivo. Ten daily injections of Flt3L, systemically expands DCs, whereas a single injection of an adenovirus vector with the Flt3L transgene (Adv-Flt3L) significantly expands DCs as soon as 2 days following intravenous (iv) injection. The increases in DC numbers, following iv injection, remains at near peak levels from day 6 through day 16. Maximal DC expansion occurs following the iv injection of 1011 virus particles, such that eight days following the iv injection of Adv-Flt3L it induced an 8 fold increase in splenic CD11c+CD11b−CD8a+ cells (immune augmenting DCs), a 6 fold increase in splenic CD11c+B220+ plasmacytoid DCs, and a 9 fold increase in splenic CD11c+CD11b+ cells (immunosuppressive DCs). The increase in DC numbers and cellularity returns to near normal levels by 22 days following iv injection of Adv-Flt3L. Despite the 2–3 fold increase in spleen cellularity and significant expansion of DCs in the spleen, blood and lungs by the iv injection of Adv-Flt3L, this route of administration has minimal to no therapeutic activity for orthrotopic mammary tumors, even when therapy is initiated against small primary tumors. In contrast, the intratumoral injection of Adv-Flt3L has significant therapeutic activity against orthrotopic mammary tumors including mice in which therapy was initiated against large tumors. Unexpectedly, no significant increase in DC infiltration of the tumor was observed following iv or intratumoral Adv-Flt3L administration. In contrast, the number of tumor infiltrating CD4+ and CD8+ cells was increased following intratumoral but not iv Adv-Flt3L administration, suggesting an association with therapeutic activity. Adv-Flt3L injection also induced DC expansion in the spleen, PB and lungs in cl-66, mammary adenocarcinoma-bearing mice. Injection of adv-Flt3L (iv or intratumoral) also stimulated the expansion of T-cells (both CD4+ and CD8+) and a type 1 T cell response as measured by qRT-PCR. Further, the therapeutic activity and increased mitogenic responses by splenic lymphoid cells to Con-A and IL-2 was not depressed in mice bearing cl-66 tumors and the effect of Adv-Flt3L, while partially associated with the Adv vector, was significantly higher in mice injected with Adv-Flt3L.