Although T lymphocytes are constitutively nonadherent cells, they undergo facultative polarity during migration and upon interaction with cells presenting cognate antigen, suggesting that cell polarity might be critical for target cell destruction. Using two-photon imaging of tumor-infiltrating T lymphocytes, we found that CD44, a receptor for extracellular matrix proteins and glycosaminoglycans, was crucial for interstitial T cell navigation and, consequently, efficient tumor cell screening. CD44 functioned as a critical regulator of intratumoral movement by stabilizing cell polarity in migrating T cells, but not during target cell interactions. Stable anterior-posterior asymmetry was maintained by CD44 independently of its extracellular domain. Instead, migratory polarity depended on the recruitment of ezrin, radixin, moesin (ERM) proteins by the intracellular domain of CD44 to the posterior cellular protrusion. Our results formally demonstrate that CD44-dependent T lymphocyte locomotion within target sites represents an essential immunologic checkpoint that determines the potency of T cell effector functions.