Cell therapies utilizing mesenchymal stem cells (MSCs) could overcome limitations of traditional treatments for reconstructing craniofacial tissues. This large-scale study explored a standardized methodology for the isolation and clinical-scale expansion of alveolar bone marrow–derived MSCs (aBMSCs). We harvested 103 alveolar bone marrow samples from 45 patients using 1 of 3 standardized methodologies. Following aBMSC isolation, cells were characterized through cell-surface marker expression and lineage-specific differentiation. Long-term cultures (> 50 population doublings [PDs]) were evaluated for transformational changes through senescence, gene expression, and karyotyping. Finally, aBMSC bone-forming potential was determined in vivo. More than 0.5 cc of bone marrow was needed to predictably isolate aBMSCs, and, regardless of methodology for harvest, cell-surface marker expression of CD73, CD90, CD105, and Stro-1 was similar for aBMSCs, being 89.8%, 98.8%, 93.8%, and 3.2%, respectively; all cells were negative for CD11b, CD19, and CD45. aBMSCs exhibited multipotency, and karyotypes were normal up to 30 PDs, with significant cell senescence beginning following 35 PDs. Additionally, aBMSCs induced ectopic bone formation following subcutaneous transplantation into mice. These findings demonstrate a predictable approach for the isolation and safe clinical-scale expansion of aBMSCs, and thus, their clinical use could be considered for craniofacial regenerative therapies.