Appropriate culture conditions cause bone marrow stem cells to differentiate into multilineage cells such as adipocytes, chondrocytes, and osteoblasts. One key factor that regulates intercellular signaling and cell differentiation is the extracellular matrix microenvironment. The composition of the extracellular matrix influences cellular functions. In the present study, we investigated the effects of a microenvironment comprising a three-dimensional apatite-fiber scaffold (AFS) that has two kinds of pores (micro- and macro pores) on proliferation and subsequent differentiation of bone marrow stem cells. Morphologic observation revealed that osteoblastic cells in the AFS were distributed primarily in the same location on the fibrous scaffold and formed bridges within micro- and macro pores. We used molecular approaches to evaluate cell proliferation and differentiation in detail. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that culturing bone marrow cells on AFS increases expression of osteocalcin (OC) mRNA compared with culture in a dish. Furthermore, cells cultured in AFS expressed type X collagen (Col X), which is a marker of hypertrophic cartilage. These data suggest that the three-dimensional microenvironment of AFS facilitates cell proliferation and differentiation, and promotes endochondral ossification of bone marrow cells.