Background: Currently, bone marrow mesenchymal stem cells (BMSCs) are commonly utilized as seed cells for the treatment of bone defects or nonunion.  However, their efficacy in bone regeneration is limited due to the severe weakening or cell death that occurs after transplantation into the body when cultured in vitro. This discrepancy is attributed to differences in the cellular living environments between in vitro and in vivo conditions. Thus, the objective of our study is to develop a proficient method for preparing BMSCs with robust survival and osteogenesis ability through a combination of hypoxic preconditioning and three-dimensional (3D) culture specifically for BMSC-based cell therapy. Methods: The primary BMSCs underwent varied hypoxic preconditioning treatments including sustained hypoxia, intermittent hypoxia, and sustained normoxia. The hanging drops technique was carried out to generate 3D microspheres. CCK8 assay, apoptosis determination assay, and live-dead staining were used to evaluate the proliferation and survival potential of BMSCs. Alkaline phosphatase, alizarin red staining, and osteogenic-related proteins were performed to assess the osteogenic differentiation abilities of BMSCs. Results: BMSCs showed stronger proliferation and survival abilities after intermittent hypoxic preconditioning and 3D culture. Moreover, cells cultured in the intermittent hypoxic preconditioning and 3D culture exhibited less apoptosis and higher osteogenic differentiation abilities, alkaline phosphatase activity, cell calcium content, and expression of osteogenic-related molecules than those cultured under conventional condition. Conclusions: The application potential of seed cells in bone regeneration can be significantly improved by subjecting BMSCs to intermittent hypoxic preconditioning and 3D spheroid culture at specific stages. These treatments promote the proliferation, survival, and osteogenic differentiation potential of BMSCs, which enhances their ability to support bone growth and regeneration.