Transfection of tumors with tumor-associated antigens (Ags) or cytokines can increase immunogenicity and slow down tumor growth. However, the effect of cotransfection with genes that encode a tumor-associated Ag, such as the tumor suppressor gene p53, and a cytokine has been rarely investigated. We report that transfection of 4T1 mammary tumor cells (p53-null) with the dendritic cell (DC) growth factor, fms-like tyrosine kinase 3 ligand (Flt3L), significantly delayed their growth in vivo, resulting in the rejection of 100% of the tumors formed by injection of tumor cells cotransfected with Flt3L and p53. Immunization with irradiated 4T1 cells transfected with Flt3L induced DC infiltration of the immunization site and significantly increased the antitumor T-cell responses. Further, immunization with irradiated 4T1 cells cotransfected with p53 and Flt3L significantly increased p53-specific immune responses, as compared to vaccination with 4T1 cells transfected with either Flt3L or p53 alone. These responses included increased activity against clone 66 (Cl-66), a sister tumor to 4T1 with high murine mutant p53 expression levels. Challenge with Cl-66 revealed that immunization with irradiated 4T1-Flt3L-p53 cells significantly slowed growth, prolonged survival, and resulted in complete remissions. Further, immunization with irradiated 4T1-Flt3L also slowed Cl-66 growth, although to a lesser extent than 4T1-Flt3L-p53. We suggest that immunization with DCs transfected with the Flt3L transgene and a tumor Ag may potentially heighten T-cell responses and therapeutic activity.