Abstract The GATA-3 transcription factor is a master regulator of cell fate determination in the mammary gland. Mice lacking expression of GATA-3 in the mammary gland fail to develop complete ductal branching or terminal end buds, while depletion of GATA-3 from adult mammary glands leads to loss of markers for luminal cell differentiation and to caspase-mediated death of luminal cells. In human breast cancer, GATA-3 expression is found in highly differentiated, estrogen receptor positive tumors with good prognosis, while loss correlates with highly metastatic tumors resistant to endocrine therapy. Regulation of GATA-3 expression is incompletely understood, though a positive-cross regulatory loop with the estrogen receptor alpha has been defined. The zinc finger protein Fliz-1 represses GATA-3 transcription in the T cell lineage, but its role in mammary cells is unknown. We hypothesize that Fliz-1 also can repress GATA-3 expression in mammary cells, leading to loss of estrogen response. To test this hypothesis, we identified a mouse mammary tumor cell line expressing high levels of Fliz1, and transfected the cells with a Fliz-1 silencing vector or negative control. Successful knockdown was confirmed by RT/PCR. Cells with decreased Fliz-1 expression showed increased expression of GATA-3 and estrogen receptor alpha mRNA relative to controls. The Fliz-1-expressing cells formed tumors that were insensitive to tamoxifen treatment when implanted in syngeneic BALB/c mice, while cells with decreased Fliz-1 expression exhibited decreased tumor growth in response to endocrine therapy. As a reciprocal experiment, we constructed a fusion gene linking Fliz-1 with a C-terminal green fluorescent protein tag. The fusion protein properly localizes to the nucleus of transfected MCF-7 (human breast cancer) and COMMA-D (mouse mammary epithelial) cells. Chromatin immunoprecipitation was used to show that Fliz1-GFP binds to an intronic regulatory region within the GATA-3 gene in COMMA-D cells. These data provide evidence supporting a role for Fliz-1 in control of GATA-3 expression in mammary cells and tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2197. doi:1538-7445.AM2012-2197