T-cell receptor stimulation by antigen bound to the major histocompatibility complex (MHC) on an antigen-presenting cell (APC) induces protein kinase C (PKC) activation and the formation of the immune synapse (IS), followed by depletion of filamentous actin (F-actin) at the central region of the IS (cIS) and the polarization of multivesicular bodies (MVB) and the microtubule-organizing center (MTOC) to the IS. These events lead to polarized exosome secretion at the IS. These exosomes are involved in several crucial immune responses such as autocrine activation-induced cell death (AICD) of T lymphocytes and citotoxicity. We analysed here how formin-like 1 β (FMNL1β), an actin cytoskeleton-regulatory protein, regulates MTOC/MVB polarization and exosome secretion at the IS in a phosphorylation-dependent manner. IS formation was associated with transient recruitment of FMNL1β to the IS, which was independent of protein kinase C δ (PKCδ). Simultaneous RNA interference of all FMNL1 isoforms prevented MTOC/MVB polarization and exosome secretion, which was restored by FMNL1β expression. However, expression of the non-phosphorylatable mutant FMNL1βS1086A did not restore neither MTOC/MVB polarization nor exosome secretion to control levels, supporting the crucial role of S1086 phosphorylation in MTOC/MVB polarization and secretion. In contrast, the phosphomimetic mutant, FMNL1βS1086D, restored MTOC/MVB polarization and exosome secretion. Conversely, FMNL1βS1086D mutant did not recover the deficient MTOC/MVB polarization occurring in a PKCδ-interfered clone, indicating that S1086 phosphorylation alone is not sufficient for MTOC/MVB polarization and exosome secretion. FMNL1 interference inhibited the depletion of F-actin at the cIS, which is necessary for MTOC/MVB polarization. FMNL1β and FMNL1βS1086D, but not FMNL1βS1086A expression, restored F-actin depletion at cIS. Thus, actin cytoskeleton reorganization at the IS underlay the effects of all these FMNL1β variants on polarized secretory traffic. Taken together, these results point out a crucial role of S1086 phosphorylation in FMNL1β activation, leading to cortical actin reorganization and subsequent control of MTOC/MVB polarization and exosome secretion.