In this study, we built a 3D perfusion system capable of applying oscillatory fluid shear stress on cells seeded in PLA scaffold. Subsequently, we studied responses of bone marrow cells to oscillatory perfusion. We first examined the responses of bone marrow cells from wild type (WT) mice and P2Y2 knockout (KO) mice to oscillatory perfusion. We found that oscillatory perfusion caused ERK1/2 phosphorylation in bone marrow cells, and the phosphorylation level in WT cells is significantly higher than that in P2Y2 KO cells. Oscillatory perfusion is also able to initiate ATP release from these cells, but there is no significant difference between WT and P2Y2 KO cells. In addition, WT bone marrow cells cultured in PLA scaffold showed higher AP activity and calcium deposition rate than P2Y2 KO bone marrow cells. Further stress relaxation tests showed that instant and long-term moduli of PLA scaffold with bone marrow cells were increased significantly after 28 d culture; and the ones with WT cells have higher moduli than those with P2Y2 KO cells. Our findings suggest that oscillatory perfusion regulates bone marrow cell responses in 3D culture conditions, and P2Y2 plays an important role in bone marrow cell mechanotransduction and osteogenic differentiation.