Chemokines promote lymphocyte motility by triggering F-actin rearrangements and inducing cellular polarization. Chemokines can also enhance cell-cell adhesion and costimulate T cells. In this study, we establish a requirement for the actin-bundling protein L-plastin (LPL) in CCR7- and sphingosine-1-phosphate- mediated T cell chemotaxis using LPL-/- mice. Disrupted motility of mature LPL-/- thymocytes manifested in vivo as diminished thymic egress. Two-photon microscopy of LPL-/- lymphocytes revealed reduced velocity and motility in lymph nodes. Defective migration resulted from defective cellular polarization following CCR7 ligation, as CCR7 did not polarize to the leading edge in chemokine-stimulated LPL-/- T cells. However, CCR7 signaling to F-actin polymerization and CCR7-mediated costimulation was intact in LPL-/- lymphocytes. The differential requirement for LPL in CCR7-induced cellular adhesion and CCR7-induced motility allowed assessment of the contribution of CCR7-mediated motility to positive selection of thymocytes and lineage commitment. Results suggest that normal motility is not required for CCR7 to function in positive selection and lineage commitment. We thus identify LPL as a molecule critical for CCR7-mediated motility but dispensable for early CCR7 signaling. The requirement for actin bundling by LPL for polarization reveals a novel mechanism of regulating actin dynamics during T cell motility.