Abstract The tumor microenvironment (TME) promotes unique epigenetic programming of stromal cell populations, including vascular endothelial cells (VEC) and pericytes, leading to differential expression of antigens that may be recognized by therapeutic T cells. These antigenic differences may be targeted immunologically via specific vaccination. We show that pericytes in human and murine renal cell carcinoma (RCC) and melanoma differentially overexpress the antigen Delta-like homolog 1 (DLK-1). DLK-1 is an EGF-like protein that functions as a Notch pathway antagonist. We demonstrate that both peptide and genetic vaccination of RENCA-bearing BALB/c mice against DLK-1 results in tumor growth inhibition and removal of DLK-1 within the TME. However, DLK-1 removal also led to a compensatory increase in DLK-2 expression (a DLK-1 homolog and NOTCH antagonist) in the murine RENCA TME. Using recombinant lentiviruses encoding murine DLK-1 and DLK-2, we show that coordinate genetic vaccination against these two antigens leads to superior therapeutic inhibition of RENCA tumor growth compared to vaccination against either DLK-1 or DLK-2 alone. The coordinate vaccination also resulted in the activation of DLK-1- and DLK-2-specific CD8+ T cells, recruitment of T cells into the TME and normalization of tumor blood vessels. We conclude that vaccination against DLK-1 and/or DLK-2 may represent an effective strategy for vascularized, solid forms of cancer, including RCC and melanoma, among others.