Here, we describe the capacity of mouse embryonic stem cells (mESCs) to differentiate into osteoblast-like cells in a three-dimensional (3D) self-assembling peptide scaffold, a synthetic nanofiber biomaterial with future applications in regenerative medicine. We have previously demonstrated that classical tissue cultures (two-dimensional) as well as 3D-systems promoted differentiation of mESCs into cells with an osteoblast-like phenotype expressing osteopontin (OPN) and collagen type I (Col I), as well as high alkaline phosphatase (Alk Phos) activity and calcium phosphate mineralization. Interestingly, in 3D self-assembling peptide scaffold cultures, the frequency of appearance of embryonic stem-cell-like colonies was substantially enhanced, suggesting that this particular 3D microenvironment promoted the generation of a stem-cell-like niche that allows the maintenance of a small pool of undifferentiated cells. We propose that the 3D system provides a unique microenvironment permissive to promote differentiation of mESCs into osteoblast-like cells while maintaining its regenerative capacity.