The osteogenic differentiation of progenitor populations allows analysis of cell functionality as well as creating a platform for investigating stem cells for bone tissue engineering. Protocols used for osteogenic differentiation of progenitor cells are often identical to those detailed for bone marrow mesenchymal stem cells, however this may be flawed due to cell populations residing in different niches and being in distinct stages of differentiation. We herein describe the individual and combined effects of known osteo-inductive agents; dexamethasone (Dex), 1,25-dihydroxyvitamin D3 (VitD3), all trans-retinoic acid (atRA), cyclic AMP (cAMP) and bone morphogenic protein 2 (BMP2) in combination with fetal bovine serum (FBS) on osteogenesis of human periosteal derived cells (hPDCs). The addition of Dex&FBS was essential for the transition of hPDCs to an ALP positive cell population. Subsequently, atRA, Dex&FBS and BMP2 were required for the expression of transcription factors governing osteogenesis and hence differentiation towards a mature osteoblast. It is also hypothesized that Dex has no direct effect on the differentiation of hPDCs, instead its effect is to augment differentiation in combination with other factors. These data provide a comprehensive assessment of known osteogenic factors, in a novel multiplex system, to evaluate their effect on progenitor cell differentiation.