There has been extensive exploration of how cells may serve as advanced therapy medicinal products to treat skeletal pathologies. Osteoblast progenitors responsible for production of extracellular matrix that is subsequently mineralized during bone formation have been characterised as a rare bone marrow subpopulation of cell culture plastic adherent cells. Conveniently, they proliferate to form single-cell derived colonies of fibroblastoid cells, termed colony forming unit fibroblasts that can subsequently differentiate to aggregates resembling small areas of cartilage or bone. However, donor heterogeneity and loss of osteogenic differentiation capacity during extended cell culture have made the discovery of reliable potency assay biomarkers difficult. Nonetheless, functional osteoblast models derived from telomerised human bone marrow stromal cells have allowed extensive comparative analysis of gene expression, microRNA, morphological phenotypes and secreted proteins. This chapter highlights numerous insights into the molecular mechanisms underpinning osteogenic differentiation of multipotent stromal cells and bone formation, discussing aspects involved in the choice of useful biomarkers for functional attributes that can be quantitively measured in osteogenic potency assays.