Directional connectivity is required to develop accurate in vitro models of the nervous system. This research investigated the interaction of murine neuronal outgrowths with asymmetric microstructured geometries to provide insights into the mechanisms governing unidirectional outgrowth bias. The structures were designed using edge-guidance and critical turning angle principles to study different prohibitive to permissive edge-guidance ratios. The different structures enable outgrowth in the permissive direction, while reducing outgrowth in the prohibitive direction. Outgrowth bias was probabilistic in nature, requiring multiple structures for effective unidirectional bias in primary hippocampal cultures at 14 days in vitro. Arrowhead structures with acute posterior corners were optimal, enabling 100% unidirectional outgrowth bias by virtue of re-routing and delay effects.