The spatial organization of the pulp cells may modify the cytodifferentiation process. The purpose of this study was to compare the two-versus three-dimensional cell culture systems for differentiation of human odontoblastic cells in vitro. Pulpal cores from freshly extracted human third molars were cultured in vitro in a perfusion device on two types of membranes: polyester membrane (twodimensional [2D] cell culture) and nylon mesh (three-dimensional [3D] cell culture). The cells were incubated with minimum essential medium containing (a) substitute serum, (b) 10% fetal calf serum (FCS), (c) 10% fetal calf serum + 2 mM β-glycerophosphate (βGP), and (d) 10% fetal calf serum + transforming growth factor (TGF)β1. Immunohistochemistry was used to evaluate the expression of collagen I, osteonectin, and nestin. Small differences were observed between 2D and 3D cell culture systems. This was particularly evident in the 10% FCS group. β-Glycerophosphate in the 3D system seems to stimulate the osteogenic cell phenotype, as a considerable induction of osteonectin is observed.