Contact inhibition of locomotion (CIL), a migratory mechanism, first introduced by Abercrombie and Heaysman in 1953 is now a fundamental driving force in developmental, repair and disease biology. Much of what we know of CIL stems from studies done on 2D substrates which are unable to provide the essential biophysical cue of fibrous extracellular matrix curvature. Here we inquired if the same rules are applicable for cells attached to and migrating persistently on suspended and aligned ECM-mimicking nanofibers. Using two elongated cell shapes (spindle attached to one fiber, and parallel attached to two fibers), we quantitate CIL rules for spindle-spindle, parallel-parallel and spindle-parallel collisions. Two approaching spindles do not repolarize upon contact but rather continue to migrate past one another. Contrastingly, approaching parallel cells establish distinct CIL, with only one cell repolarizing upon contact followed by migration of both cells as a cohesive unit in the repolarization direction. Interestingly, for the case of spindle and parallel cell collision, we find the parallel cell to shift the morphology to that of spindle and continue persistent movement without repolarization. To account for effect of cell speed, we also quantitate CIL collisions between daughter and non-dividing cells. While spindle-spindle collisions result in cells still walking by, for parallel-parallel collisions, we capture rare events of a daughter cell pushing the non-dividing cell. With increasing population numbers, we observe formation of cell streams that collapse into spheroids. Single cells are able to invade along fibers from the spheroids and are then subject to same CIL conditions, thus providing a platform with cyclic CIL. The presented coupling of experimental and analytical framework provides new insights in contextually relevant CIL and predictive capabilities in cell migration decision steps. / Master of Science / Contact inhibition of locomotion (CIL) is a migratory process that can lead to a change in migration direction through protrusion inhibition of single cells. First described in 1953, the traditional model of CIL shows that on a 2D substrate, two migrating cells experience a decrease in protrusive behavior upon contacting each other, followed by repolarization, and migration away from one another. However, a cell's extracellular matrix (ECM) is fibrous in nature, and how cells maintain standard CIL rules in fibrous environments remains unclear. Here, using suspended, aligned nanofibers created using a non-electrospinning Spinneret based Tunable Engineered Parameters (STEP) method, we investigate CIL decision steps of two fibroblast cells approaching each other in two shapes: spindle cells attached to single fibers, and parallel cells attached to two fibers. Most spindle cells approaching each other do not switch direction upon contact, but rather continue to migrate past each other, termed a walk past. Contrastingly, approaching parallel cells display unique CIL whereby only one cell repolarizes and reverses its migration direction. Subsequently, both cells remain in contact while migrating in the repolarization direction. Interestingly, we report that both spindle and parallel CIL are also affected by speed post cell division. Altogether, for the first time, we introduce a platform to understand cell shape driven CIL geometrical rules in ECM mimicking environments.