Musculoskeletal tissue-engineering strategies have recently focused on the use of biomaterial scaffolds capable of guiding growth and organization of mesenchymal stem cells (MSCs), which are precursors for connective tissues. This chapter describes the methods for culturing MSCs on micropatterned polydimethylsiloxane (PDMS) substrates. MSCs are isolated from bone marrow biopsies and subcultivated before plating onto PDMS substrates. Micropatterned substrates are fabricated by casting PDMS on AZ P4620 photoresist molds. Prior to plating cells, substrates are cleaned, sterilized, and coated with fibronectin. Micropatterned growth surfaces are a useful research tool enabling the study of cell morphology and alignment in response to substrate geometry. Understanding MSC response to surface topography will assist in the design of improved scaffolds for connective-tissue repair.