We have previously demonstrated that osteogenic differentiation is inhibited and angiogenic expression is enhanced in murine preosteoblasts (MC3T3-E1) cultured on three-dimensional (3D) poly-L-lactide-co-glycolide (PLGA) scaffolds when compared to two-dimensional (2D) PLGA films. In the present work we investigated the role of the extracellular signal-related kinase 1/2 (ERK1/2) pathway in modulating osteogenic and angiogenic differentiation in 2D and 3D systems made of two distinct biomaterials-type I collagen and PLGA. The addition of a third dimension, regardless of biomaterials, substantially increased ERK1/2 activation as demonstrated by an increase in phosphorylated ERK1/2. Western blot analysis showed significant increases in phosphorylation of ERK1/2 in cells grown in 3D versus 2D cultures at day 4 (5- and 7.7-fold increases 3D vs. 2D in collagens and PLGA, respectively) and day 7 (4.7- and 4.6-fold increases 3D vs. 2D in collagen and PLGA, respectively). Using an ERK-specific inhibitor, PD 98059, we established a correlation between ERK activation and inhibited osteogenic differentiation. Inhibition of ERK activation in 3D cultures significantly enhanced osteogenic differentiation. It in fact restored osteogenic differentiation to a level equal to that of 2D cultured cells. Inhibition of ERK1/2, however, showed little effect on angiogenic gene expression, indicating that two distinct mechanisms are involved in osteogenic and angiogenic differentiation. Taken together, these results allow us to report a mechanistic model in MC3T3-E1 cells in which distinct activation of ERK1/2 in 3D culture has an inhibitory effect on osteogenic differentiation.