In the present work, we studied the differentiation capacity of mouse embryonic stem cells (mESCs) and mouse embryonic fibroblasts (MEFs) to differentiate into osteoblast-like cells in a 3-dimensional (3D) self-assembling peptide scaffold, a synthetic nanofiber biomaterial with potential applications in regenerative medicine. We demonstrated that 2D and 3D systems promoted differentiation of mESCs into cells with an osteoblast-like phenotype consisting of osteopontin and collagen I marker expression, as well as high alkaline phosphatase (ALP) activity and calcium phosphate deposits. In 3D cultures the frequency of appearance of embryonic stem cell-like colonies was substantially greater, suggesting that the 3D microenvironment promoted the generation of a stem cell-like niche that allows undifferentiated stem cell maintenance. On the other hand, after MEFs were cultured in the 3D system with their regular growth medium, but not in the 2D system, they expressed osteopontin, up-regulated metalloproteinase activities, and acquired a distinct phenotype consisting of small, elongated cells with remaining mitotic activity. Furthermore, only 3D MEF cultures underwent osteoblast differentiation after osteogenic induction, based on matrix mineralization, collagen I synthesis, ALP activity, and expression of the osteoblast transcription factor Runx2, suggesting that the 3D environment promotes differentiation of MEFs into osteoblast-like cells. We propose that the 3D system provides a unique microenvironment that promotes differentiation of mESCs and MEFs into osteoblast-like cells.