Actin dynamics is essential for T cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis, or surface receptor trafficking. However, in cofilin1 mutant mice impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double positive thymocytes towards CD4 and CD8 single positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration dependent cell differentiation.