Osteosarcoma (OS) is a highly heterogeneous and aggressive bone malignancy whose complexity is strongly influenced by its Tumor Microenvironment (TME). Within this niche, Mesenchymal Stem Cells (MSCs) play a pivotal role in tumor progression by undergoing phenotypic and functional reprogramming under tumor-derived cues, acquiring Cancer-Associated Fibroblast (CAF)-like features that promote proliferation, invasion, and immune evasion. The Extracellular Matrix (ECM), once regarded as a passive structural element, is now recognized as an active regulator of tumor behavior, acting as a reservoir of signaling molecules and a modulator of cell fate. However, the molecular crosstalk between OS cells, MSCs, and the ECM remains poorly understood, largely due to the limitations of conventional two-dimensional models. In this study, we established a three-dimensional (3D) bone-mimetic model of osteosarcoma (mOS-3D) by co-culturing human OS cells and MSCs within a hydroxyapatite–collagen (MgHA/Coll) scaffold that recapitulates the biochemical and structural features of native bone ECM. This in vitro platform reproduces key aspects of the OS microenvironment, enabling the investigation of tumor–stroma interactions and their impact on stemness, stromal activation, and ECM remodeling. The mOS-3D model provides a physiologically relevant and tunable system for studying the cellular mechanisms driving OS progression and offers a promising preclinical tool to explore therapeutic strategies targeting the TME.