Understanding how cells assemble internal structures is central to cell biology. In Trypanosoma brucei, the flagellar pocket (FP) is essential for nutrient uptake, and immune evasion, and its formation depends on a cytoskeletal structure called the flagellar pocket collar (FPC). However, the mechanisms underlying FPC assembly remain poorly understood. In this study, we used cutting-edge ultrastructure expansion microscopy (U-ExM) to investigate FPC biogenesis in T. brucei. We mapped the formation of the proximal part of the new microtubule quartet (nMtQ) alongside flagellum growth, providing new insights into its assembly. Additionally, we tracked the localization dynamics of key structural proteins-BILBO1, MORN1, and BILBO2-during the biogenesis of the FPC and the hook complex (HC). Notably, we identified two previously undetected structures: the proFPC and the transient FPC-interconnecting fiber (FPC-IF), both of which appear to play crucial roles in linking and organizing cellular components during cell division. By uncovering these novel aspects of FPC biogenesis, our study significantly advances the understanding of cytoskeletal organization in trypanosomes and opens new avenues for exploring the functional significance of these structures.Read less <