Bone formation and regeneration are essential for maintaining and repairing the skeleton, with osteoblastogenesis as the key process where mesenchymal stem cells (MSCs) differentiate into osteoblasts. This complex process is regulated by various signaling pathways, which promote osteoblast differentiation, proliferation, and extracellular matrix mineralization in response to fractures or bone loss. This process requires a series of cellular events and is regulated by growth factors, cytokines, and mechanical stimuli. With large defects or pathological states, natural regeneration may be insufficient, requiring therapeutic interventions. Biomaterial scaffolds, gene therapy, stem cell therapy, and tissue engineering have greatly enhanced bone repair. The combination of MSC with bioactive agents has shown promise in promoting osteoblast formation and accelerating bone regeneration. Recent advancements in nanotechnology and 3D bioprinting have led to innovative methods for creating biomimetic scaffolds that support bone tissue formation through structural and biochemical cues. The research aims to optimize regenerative approaches, improve scaffold biocompatibility, and identify molecular targets that could greatly improve patient outcomes in orthopedics and reconstructive medicine. This chapter discusses microenvironments supporting osteogenesis, the long-term safety of new treatments, and the potential of targeting a transcription factor, and its downstream pathways to enhance bone regeneration.