Vitronectin-derived cell culture substrates and methods of using the same for culturing pluripotent stem cells are presented. Also provided herein are defined culture systems for maintaining human pluripotent stem cells in a substantially undifferentiated state, where the defined culture system comprises human pluripotent stem cells, a defined culture medium, and at least one polypeptide selected from the group consisting of residues 43 to 378 of SEQ ID NO:1 and residues 45 to 378 of SEQ ID NO:1, and where the defined culture medium comprises insulin, selenium, transferrin, L-ascorbic acid, FGF2, DMEM/F12, NaHCO3, and one of TGFβ and NODAL.