Tissue engineering provides a valuable tool for in vitro investigation of complex in vivo environments. A particular application of tissue-engineered in vitro platforms in neuroscience and regenerative medicine is the fabrication of controlled microenvironments for the study of axon guidance, with the goal of informing strategies to overcome nerve injury. The innovative design of tissue-engineered scaffolds that incorporate multiple guidance cues and cell types into various environments is advancing the understanding of how neurons integrate guidance information to make growth decisions. This review focuses on recent strategies that present neurons with multiple cues with micro- and nanoscale resolution in order to study the interactions between neurons and their local environment during axon guidance.