The implementation of micro- and nanotechnologies to biomaterials constitutes a unique platform to improve our understanding on microenvironmental regulation of stem cell functions. In the recent years, various methods have been developed for the fabrication of micro- and nanopatterned polymeric culture substrates, and many of these novel surfaces are opening possibilities for new applications. Here, we provide procedures for creating nanoscale topographic features on films of poly(lactic acid), a biodegradable polymer frequently used for the fabrication of tissue engineering scaffolds. In addition, we provide methods to assess the growth and differentiation of mesenchymal stem cells cultured on the substrates.