After the clinical insertion of a bone biomaterial, the surrounding osteoblasts would migrate and attach to the implant surface and foster a microenvironment that largely determines the differentiation fate of the comigrated mesenchymal stem cells. Whether the fostered microenvironment is suitable for osteogenic differentiation of mesenchymal stem cells is critical for the subsequent osseointegration. In this study, we determined (1) how the spherical or rod-shaped hydroxyapatite nanoparticles (nHA) incorporated poly(ɛ-caprolactone) (PCL) films (PCL-spherical nHA, PCL-rod nHA) interact with primary human osteoblasts (HOBs); (2) how the microenvironment rendered by their interaction affects osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs). HOBs were seeded on PCL, PCL-spherical nHA, and PCL-rod nHA films, respectively. When cultured alone, the HOBs on PCL-rod nHA films showed most efficient osteoblastic differentiation compared with those on PCL or PCL-spherical nHA films. When cocultured with ASCs in an indirect coculture system, only the HOBs on PCL-rod nHA films up-regulated the gene expression of Runx2, bone sialoprotein, and osteocalcin of ASCs. Additionally, the HOBs on PCL-rod nHA films showed significant up-regulation of bone morphogenic protein 2 gene and protein expression and induced highest phosphorylated Smad1/5 protein level in ASCs. Treatment of the coculture medium with bone morphogenic protein 2 inhibitor (Noggin) largely abolished the osteogenic differentiation of the ASCs induced by the HOBs on PCL-rod nHA films. In conclusion, HOBs can not only best display their osteoblastic phenotype by culturing on PCL-rod nHA films but also render an optimal osteogenic niche for the differentiation of stem cells.