In order to mimic in vivo topography of the native tissue created by extracellular matrix (ECM) components, which make up all soft tissues, the surface features of each biomaterial should be considered as a nanodimensional structure. In this study, an artificial ECM was designed to mimic the nanostructured topography created by ECM components in native tissue. The proliferation and differentiation of mesenchymal stem cells (MSCs) was investigated in a three dimensional (3-D) network of nanofibers formed by the self-assembly of peptide amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH₂ terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing MSC suspensions in a media with dilute aqueous solution of PA. The attachment, proliferation and osteogenic differentiation of MSCs were influenced by the self-assembled PA nanofibers as the cell scaffold and the values were significantly high compared with those in the static culture (2-D tissue culture plate).