Bone is the most frequent site of metastasis for patients with advanced breast cancer. Despite its clinical impact, bone metastases remain incurable with current treatments primarily aimed at slowing progression and alleviating skeletal symptoms. Most previous research in the field has focused on studying late-stage metastases, which are characterized by a vicious cycle of bone degradation. However, recent studies revealed that disseminated breast cancer cells initially colonize regions of new bone formation known as osteogenic niches where they interact with mesenchymal stem cells (MSCs) and the bone extracellular matrix (ECM). While MSCs are known to regulate metastasis and dormancy through direct cell-cell interactions, the role of the bone ECM remains underappreciated. Clinically, reduced bone matrix mineral density correlates with an increased risk of developing bone metastases. However, the mechanisms linking reduced mineral content to metastatic progression remain poorly understood. Thus, the overall goal of this dissertation was to investigate whether bone matrix mineralization regulates MSC phenotype and how this crosstalk influences breast cancer metastasis. To address these questions, two bone-mimetic engineered models enabling systematic control over matrix mineral content were employed. These models were used to examine (1) the interactions between MSCs and the bone ECM, and (2) the influence of systemic factors on MSC-matrix crosstalk, with a focus on potential synergistic effects. More specifically, this work explored how breast cancer-derived factors influence MSC phenotype and whether MSC responses were dependent on matrix mineralization. This doctoral work also examined the role of bacterial-derived outer membrane vesicles (OMVs), which arrive in bone from the gut microbiome, in regulating MSC-bone matrix crosstalk. Collectively, the findings from this dissertation demonstrate that mineralized bone matrix alone can direct MSC phenotype, which in turn regulates breast cancer cell growth both in vitro and in vivo. Additionally, systemic factors such as breast cancer-derived factors and OMVs significantly alter MSC-matrix interactions, with functional consequences on breast cancer cell growth. While further studies are needed to fully elucidate the underlying mechanisms, this work provides critical new insights into how mineralized bone matrix and systemic factors jointly shape the early metastatic bone microenvironment. These findings advance our understanding of breast cancer bone metastasis and highlight novel areas for therapeutic intervention.