Purpose: Glioblastoma multiforme (GBM) is a deadly primary brain tumor. Clinical trials for GBM using dendritic cell (DC) vaccination resulted in antitumor immune responses. Herein, we tested the hypothesis that combining in situ (intratumoral) Ad-Flt3L/Ad-TK-mediated gene therapy with DC vaccination would increase therapeutic efficacy and antitumor immunity. Experimental Design: We first assessed the immunogenicity of tumor lysates generated by Ad-TK (+GCV), temozolomide (TMZ), or freeze/thawing cycles (FTC) in a syngeneic brain tumor model. We also assessed phenotypic markers, cytokine release, and phagocytosis of bone marrow-derived DCs generated by fms-like tyrosine kinase 3 ligand (Flt3L) + IL-6 or by granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin (IL) 4. Inhibition of tumor progression and production of anti-GBM antibodies was assessed following vaccination with (i) tumor cell lysates, (ii) DCs generated with either Flt3L/IL-6 or GM-CSF/IL-4 loaded with either Ad-TK/GCV-, TMZ-, or FTC-generated tumor lysates, or (iii) DCs in combination with in situ Ad-Flt3L/Ad-TK gene therapy. Results: DCs loaded with tumor cell lysates generated with either Ad-TK/GCV or TMZ led to increased levels of phagocytosis, therapeutic efficacy, and humoral immune response. In situ immunogene therapy in combination with DC vaccination led to brain tumor regression and long-term survival in about 90% of animals, a significant increase when compared with either therapy alone. Conclusions: Our results indicate that modifying the tumor microenvironment using intratumoral Ad-Flt3L/Ad-TK-mediated gene therapy potentiates therapeutic efficacy and antitumor immunity induced by DC vaccination. These data support novel phase I clinical trials to assess the safety and efficacy of this combined approach. ©2011 AACR.