As o ur s cienti c c omprehension adv ances, o ur s earch to u nderstand t he w orld re quires s cientists to i nvestigate smaller a nd smaller features. In t he eld of tissue engineering this is no di erent. e constant endeavor for new knowledge drives the development of methods of study involving cells, and/ or biomaterials to heal illness and improve longevity (Khademhosseini et al., 2006). However, it is necessary to fully understand the current applications to enable the development of future techniques. ese applications focus on the combination of biological and engineering techniques to mimic the structures of the human body like the extracellular matrix (ECM) in order to rebuild tissue function (Khademhosseini et a l., 2006; Nelson a nd Tien, 2006). is is a very di cult task. e ECM is a h ighly heterogeneous structure composed of many biomolecules with di erent chemical features that create di erent microenvironments (Nelson and Tien, 2006). Cells in these di erent environments respond, grow, and (in the case of stem cells) develop di erently (Bettinger e t a l., 2009). ese cells receive d i erent migration, proliferation, and adhesion signals from the ECM microenvironment around them. It has been shown in vitro that micro and nanoscale patterned substrates can be used to produce structural and chemical cues to cause changes in cell behavior (Khademhosseini et al., 2006; Nelson and Tien, 2006; Bettinger et a l., 2009). St ill, researchers lack t he ability to re create t hese unique matrices synthetically and can only re create c hemical or s tructural m icroenvironments m imicking a f ew nat ural c ues (Nelson a nd Tien, 2 006). is c hapter w ill f ocus o n t wo-dimensional m icro a nd na nopatterning o f b iomimetic substrates. e he terogeneity o f t he m icroenvironment i s i mportant i n re gulating c ellular f unction (Nelson and Tien, 2006; Bettinger et al., 2009). Modi cation of natural and synthetic biomimetic substrates is necessary to tailor them to the speci c application of interest (i.e., cell study, treatment, etc.).