Epithelial cells have a highly polarized cytoskeleton, in which microvilli, finger-like protrusions with filamentous actin (F-actin) cores, protrude only from the apical side. Ezrin, a plasma-membrane-F-actin crosslinker, is highly enriched in microvilli, and required for their presence in some cultured cell-lines. Numerous binding partners of ezrin are known, including ezrin-binding phosphoprotein 50 (EBP50) and CD44, but no systematic analysis of ezrin-binding partners in epithelial cells has been undertaken. To better understand how ezrin works, we conducted a proteomic profiling of ezrin immunoprecipitates and identified 12 novel and 7 previously known interactors. The N- and Ctermini of ezrin interact forming a dormant molecule, and this autoinhibition is released in a process dependent upon phosphorylation of a critical C-terminal residue, threonine-567, so we can control it through mutation of T567. By quantitatively comparing immunoprecipitates from open vs. closed ezrin mutants, I found that different ezrin binding partners respond differently to changes in ezrin autoinhibition. In the course of my studies, I made the surprising observation that open ezrin variants lost their polarized distribution within microvilli, while they remained on the plasma membrane. This implied that a local cue, operating just in the apical domain, controls the normal opening of ezrin. A likely candidate was the hypothetical T567 kinase. Among the 12 novel ezrin-binding proteins, I noticed 2 potential T567 kinases, LOK and SLK, and confirmed that their presence was required for T567 phosphorylation and microvilli in two cell-lines. In additional experiments, I determined that their apically-localized activity was required for ezrin's apical localization, which in turn, was required for the de novo presence of microvilli. Thus, a critical aspect of epithelial cytoskeletal polarity is the control of ezrin's partner interactions through the regulated release of autoinhibition mediated by apically polarized T567 phosphorylation.