Here we report the design of a unique matrix, assembled from engineered M13 phage bionanofibers with specific cues of nanotopographies and versatile signal peptides to simulate native niche for directing the fate of induced pluripotent stem cells (iPSCs). By independently varying the peptide sequences and nanotopographies, we find that the resident iPSCs on the phage matrix are first differentiated into mesenchymal progenitor cells (MPCs), which are further differentiated into osteoblasts in the absence of osteogenic supplements due to the elongation induced by phage nanofibers. The phage-based matrix represents not only a biomimetic stem cell niche enabling independently varying biochemical and biophysical cues in one system but also a substrate for generating a safe and efficient cell source for tissue engineering.