In addition to providing maneuverability, electrospun nanofibrous meshes can make excellent supports for constructing flexible cell sheets to regulate cell behavior by nanofiber features. With the target ofbone regeneration, herein composite nanofibers with two different fiber arrangements (nestlike, random) were electrospun from a blend solution containing poly(L-lactide) (PLLA) and gelatin (1:1 in weight ratio). Unlike the non-woven morphology in a random nanofibrous mesh, PLLA/gelatin composite nanofibers in the nestlike nanofibrous mesh displayed both non-woven and parallel morphologies. Both kinds of nanofibrous mesh were similar to 50 Tau mu m thick as-prepared, and shrank to 30 tm after seeding with bone mesenchymal stromal cells (BMSCs). After 7 days of in vitro culture, cell sheets could form on both meshes (CSM) and on the culture plate. It was found that application of nanofibrous mesh promoted the osteogenic differentiation of BMSC sheets compared with the control. The nestlike mesh displayed slight superiority over the random mesh in enhancing osteogenic differentiation, but their different fiber arrangements did not cause much difference in cell proliferation. Three-dimensional multi-layered CSM constructs were built by stacking four mono-layered CSMs together. The CSM constructs (based on a nestlike or random nanofibrous mesh) were incubated in vitro for 3 days before being implanted into rat cranial defects. In comparison with the control group, there was significant formation of new calcified bone in both CSM construct-filled groups at 12 weeks' post-operation. The nestlike group showed slightly better bone healing (based on both qualitative and quantitative analysis) than the random group, while showing insignificant differences. We showed that the concept of using a three-dimensional multi-layered CSM construct in enhancing bone regeneration was feasible. Future studies should take more nanofiber features (e.g. bioactive components) into account to further enhance osteogenesis.