Mesenchymal stem cells (MSCs) are crucial for skeletal development, homeostasis, and repair, primarily through their differentiation into osteoblasts and other skeletal lineage cells. Key signaling pathways, including Wnt, TGF-β/BMP, PTH, Hedgehog, and IGF, act as critical regulators of MSC osteogenesis, playing pivotal roles in maintaining bone homeostasis and facilitating regeneration. These pathways interact in distinct ways at various stages of bone development, mineralization, and remodeling. This review provides an overview of the molecular mechanisms by which these pathways regulate MSC osteogenesis, their influence on bone tissue formation, and their implications in bone diseases and therapeutic strategies. Additionally, we explore the potential applications of these pathways in bone tissue engineering, with a particular focus on promoting the use of MSCs as seed cells for bone defect repair. Ultimately, this review aims to highlight potential avenues for advancing bone biology research, treating bone disorders, and enhancing regenerative medicine.