Cell adhesion to material surfaces regulates host responses to implanted biomaterials and the performance of cell arrays and biotechnological cell culture supports. Therefore, the engineering of substrates that control cell adhesive interactions is critical to the development of bio-interactive interfaces and biotechnological culture supports. We describe the application of advanced fabrication techniques to engineer substrates with well defined chemistry and topography to manipulate cell adhesive interactions. Microcontact printing of self-assembled monolayers and hot embossing imprint lithography approaches were integrated to manipulate focal adhesion assembly, cell adhesion, and cellular spreading and alignment. These micro- and nanopatterned substrates provide useful tools for the analyses of structure-function relationships in adhesive interactions.