Abstract: Immune evasion is a hallmark of cancer, and immune checkpoint blockade has shown success in harnessing anti-tumor T cells to treat cancer. B7-H4, a B7 family inhibitor of T cell activity, is expressed on immune cells and is highly elevated in human tumors. Further, B7-H4 overexpression in human cancers correlates with decreased infiltrating lymphocytes and poor prognosis. To investigate the role of B7-H4 in tumor-immune interactions, we used transplantable 4T1 murine mammary carcinoma cells in syngeneic hosts. B7-H4 knockout host mice displayed augmented anti-tumor activity against 4T1 cells; however, tumors grew similarly between B7-H4 knockout and wild-type hosts. We provide evidence that this is due to the dual inhibition of both T cells and immunosuppressive myeloid cells by B7-H4 in the 4T1 model. In contrast, when a highly immunogenic 4T1 derivative (4T1-12B) was used, B7-H4 knockout mice exhibited significant tumor reduction correlating with greater tumor-associated T cell responses. Moreover, B7-H4-deficiency synergized with the chemotherapeutic agent, gemcitabine, further slowing tumor growth, and in some cases, eradicating tumors and generating anti-tumor memory T cells. Collectively, these findings show that inhibition of host B7-H4 can enhance anti-tumor T cell immunity particularly against immunogenic tumors, and suggest that B7-H4 blockade may be combined with other anti-cancer therapies to treat human cancers.