Accurate assembly of kinetochores is vital for precise chromosome segregation, as deviations may cause genetic defects. Therefore, understanding kinetochore assembly mechanisms is a longstanding goal in cell division research. Prior research demonstrated interactions between purified kinetochore proteins in vitro. However, it remains uncertain whether these interactions occur similarly in human cells.To address this, I systematically tested different steps of kinetochore assembly in human mitotic cell extracts using microscopy-based reconstruction assays. I found that cellular extracts do not facilitate de novo assembly of kinetochore complexes. Although some interactions, like those involving CENP-A-nucleosomes and CENP-C, are permitted, recruitment of the main kinetochore microtubule binder, the Ndc80 complex, to both its receptors CENP-T and CENP-C, is obstructed. My findings suggest that yet-unknown molecular mechanisms prevent interactions between kinetochore proteins in the cytoplasm of human cells. To investigate possible regulatory mechanisms, I focused on interactions between Ndc80 and CENP-T. A notable difference between the kinetochore-localized and soluble CENP-T is evident in their molecular density: CENP-T is present in multiple copies at the kinetochore, but it exists as monomers in the cytoplasm. Consequently, I compared the kinetics of Ndc80 binding to clustered versus monomeric CENP-T. Using single-molecule fluorescence techniques in vitro and purified proteins, I discovered that although both CENP-T forms bind Ndc80 with similar kinetics, their unbinding differs. I observed a time-dependent increase in the retention of Ndc80 molecules, which we termed "binding site maturation". It was significantly accelerated in clustered CENP-T. Such tunable acceleration of slowly maturing binding sites by a high molecular-density environment may represent a mechanism to assist kinetochore assembly. In line with my findings, our collaborators demonstrated the assembly of kinetochore-like particles on CENP-T clusters, as opposed to monomers, in HeLa cells. Using in vitro assays, I found that isolated kinetochore-like particles function similarly to authentic kinetochores: bind microtubules and support movements at microtubule tips. The formation of such artificial kinetochores on CENP-T clusters supports the proposed density-dependent activation mechanism for kinetochore assembly. This mechanism may play role in facilitating stable interactions among kinetochore components, and for safeguarding against the spontaneous formation of harmful kinetochore subcomplexes in the cytoplasm.