Abstract Breast cancer preferentially metastasizes to the bone, where the five-year relative survival rate falls from 90% to <10%. Although the precise mechanism underlying preferential metastasis is unknown, the bone likely provides a hospitable environment that both attracts breast cancer cells and allows them to colonize and grow. Besides affects osteoblast and osteoclast properties, we have evidence that metastatic breast cancer cells further create a unique bone niche by orchestrating extensive crosstalk with osteoblasts that may be involved in creating a niche permissive for breast cancer cell dormancy. MC3T3-E1 murine osteoblasts were grown to confluence, then MDA-MB-231 human breast cancer cells added at a ratio of 1 breast cancer cell to 10 osteoblasts. Co-cultures were subsequently fixed and stained for Cx43 expression. Next, MC3T3-E1 cells and MDA-MB-231 cells were grown to confluence. Culture supernatants were removed and processed through a series of ultra-centrifugation, purification, and concentration steps to purify for exosomes. Finally, MC3T3-E1 cells were grown to various stages of maturity: 4 (growth) and 10 days (early differentiation) and incubated with conditioned medium from human MDA-MB-231 cells. qPCR was used to assay for changes in osteoblast-derived inflammatory cytokine expression. Expression of the gap junction protein Cx43 was localized between breast cancer cells and osteoblasts. Both MC3T3-E1 and MDA-MB-231 cells expressed exosomes, which were visualized using a transmission electron microscope. Osteoblast-derived cytokines increased in the presence of MDA-MB-231 breast cancer cell conditioned medium included MCP-1 (CCL2), LIX (CXCL5), LIF, and NFATc2. Overall, these data suggest that suggest there is extensive crosstalk directly between osteoblasts and breast cancer cells, and that osteoblasts are an important source of cytokines in breast cancer bone metastasis. Thus, these findings implicate the bone microenvironment and cancer cell manipulation thereof in orchestrating metastatic tumor cell dormancy, colonization, and survival. Supported in part by the National Institutes of Health; 1RC1 CA146381, 1R01NS06994, P50 CA083639 for FCM; Ruth L. Kirschstein Institutional Research Service Award (NRSA) T32 CA079448 for KMB. Citation Format: Karen M. Bussard, Frank C. Marini. Crosstalk between breast cancer and stromal cells occurs via exosomes and gap junctions in bone metastatic breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1625. doi:10.1158/1538-7445.AM2013-1625