Osteogenesis is one of the principal components of periodontal tissue development as well as regeneration. As pluripotent cells with unlimited proliferative potential and differentiation ability to all germ layer representatives, embryonic stem cells also hold the promise to become a cell source in bone tissue engineering. Our aim was to investigate osteogenic differentiation potential of human embryonic stem cells (hESCs) under the inductive influence of human periodontal ligament fibroblast (hPDLF) monolayers. After being expanded and characterized morphologically and immunohistochemically, hESCs (HUES-9) were cocultured with hPDLFs for 28 days. Two groups were established: (i) osteogenic induction group with ascorbic acid, beta-glycerophosphate, and dexamethasone containing hESC differentiation medium; and (ii) spontaneous differentiation group cultured in hESC differentiation medium. Morphological shift in cells was analyzed under an inverted microscope, and immunohistochemistry was performed on fixed specimens at days 1 and 28 using antibodies against alkaline phosphatase, osteonectin, osteopontin, bone sialoprotein (BSP), and osteocalcin (OSC). Reverse transcription-polymerase chain reaction was utilized for the detection of octameric binding protein-4, BSP, and OSC expression at mRNA level. Mineralization was assessed using alizarin red, and the surface topology shift in colonies was demonstrated with scanning electron microscopy. Results indicate the feasibility of osteogenic differentiation of hESCs in coculture, and suggest a role of periodontal ligament fibroblasts in their differentiation patterns. Advances in the field could allow for potential utilization of hESCs in periodontal tissue engineering applications involving regeneration of bone in periodontal compartment lost as a result of destructive periodontal diseases.