Cross-talks between actin and microtubules underly various cellular processes. Using immune synapse model and via cytoskeleton dynamics analyses, here, we demonstrate that CD99 is critical for the actin and microtubule cross-talks and the coordinated reorganization, mediating their physical linkage near the microtubule-organizing center, lamella, and cell cortex. Via the transmembrane and cytoplasmic domains, CD99 interacts with MyoIIA and IQGAP1, and facilitates interaction between these two partners and their interactions with microtubules at the three linkage sites.