Characterization of directed differentiation protocols is a prerequisite for understanding embryonic stem cell behavior, as they represent an important source for cell-based regenerative therapies. Studies have investigated the osteogenic potential of human embryonic stem cells (HESCs), building upon those using pre-osteoblastic cells, however no consensus exists as to whether differentiating HESCs behave in a similar manner to the traditionally used osteoblastic progenitors. Thus, the aim of the current investigation was to define the gene expression pattern of osteoblastic differentiating HESCs, treated with ascorbic acid phosphate, beta-glycerophosphate and dexamethasone over a 25 day period. Characterization of the gene expression dynamics revealed a phasic pattern of bone-associated protein synthesis. Collagen type I and osteopontin were initially expressed in proliferating immature cells, whereas osterix was up-regulated at the end of active cellular proliferation. Subsequently, mineralization-associated proteins, bone sialoprotein and osteocalcin were detected. In light of this dynamic expression pattern, we concluded that two distinguishable phases occurred during osteogenic HESC differentiation; first, cellular proliferation and secretion of a pre-maturational matrix, and second the appearance of osteoprogenitors with characteristic extracellular matrix synthesis. Establishment of this model provided the foundation of a time-frame for the additional supplementation with growth factors, BMP2 and VEGF. BMP2 induced the expression of principle osteogenic factors, such as osterix, bone sialoprotein and osteocalcin, whereas VEGF had the converse effect on the gene expression pattern.