Cytokinesis, the final phase of cell division, physically separates one cell into two daughter cells. Its spatiotemporal control is crucial for metazoan development, and failures in the cytokinetic machinery often are associated with the formation of cancer. During cytokinesis, an actomyosin contractile ring (CR) constricts and transitions into the midbody ring (MR), a structure that forms around the midbody as it matures from the midzone microtubules (MTs) that become compacted during CR closure. In Drosophila S2 cells, we recently showed that the conserved scaffold protein Anillin promotes the transition from CR to MR. During this maturation process, the Citron kinase Sticky retains Anillin’s N-terminus for incorporation into the MR, while the septin Peanut acts on the C-terminus of Anillin to remove excess Anillin and membrane via shedding. We found that Anillin-depleted S2 cells not only fail to form a proper MR, but also are deficient in forming a constricted zone of MTs at the midbody. Interestingly, Sticky-depleted cells, which also fail MR formation and cytokinesis, exhibit no earlier MT defects at the midbody. Hence, MR formation (Sticky-dependent) and midbody maturation (Sticky-independent) are two separable Anillin-dependent events, which we aim to further dissect in this study. Using a diverse panel of Sticky and Anillin truncations combined with live imaging and RNAi of endogenous proteins, we are currently defining the contributions of specific domains of Sticky and Anillin required for their normal CR localization and retention at the MR. Because shedding depends on Anillin, and not on Sticky, removal of Sticky likely occurs via its association with Anillin. Surprisingly, we found that Sticky-ΔRBD-FP is shed, but fails to retain Anillin at the MR, suggesting that Sticky's association with Anillin and its ability to form a stable MR may be separate events. Also, our preliminary data from immunoblotting suggest that the very N-terminus of Anillin is sufficient to pull-down Sticky from S2 cell lysate. Taken together, this work will strengthen our understanding about how the cell cortex is organized and re-organized in a mechanistic way to ensure the robustness of cytokinesis.