Cross-regulation of RUNX1 expression by RUNX3 plays a critical role in regulating proliferation of human B cells infected with Epstein-Barr virus (EBV). When EBV infection induces RUNX3, the consequent reduction in RUNX1 levels is required for the ensuing cell proliferation because forced expression of RUNX1 in an EBV lymphoblastoid cell line prevented cell proliferation. The TEL-RUNX1 fusion gene from acute B-lymphocytic leukemia retains almost all of the RUNX1 sequence but does not prevent B-cell proliferation in the same assay. B-cell maturation antigen (BCMA) was found to be induced by conditionally expressed RUNX3 in a lymphoma cell line. Chromatin immunoprecipitation assays confirmed that RUNX3 binds to the RUNX1 promoter in a lymphoblastoid cell line and a Burkitt's lymphoma cell line. The TLE binding VWRPY sequence from the C terminus of RUNX3 was found to be required for repression of the RUNX1 P1 promoter in a B-lymphoma cell line. The mechanism of repression in B-cell lines most likely involves recruitment of corepressor TLE3 or TLE4 to the RUNX1 promoter. The results demonstrate the importance of RUNX3-mediated repression of RUNX1 for EBV-driven B-cell proliferation and identify functional differences between human RUNX family proteins.