Accurate segregation of duplicated chromosomes (mitosis) relies on formation and correct dynamics of a robust mitotic spindle. This fundamental subcellular structure, present in all eukaryotic cells, is composed of microtubules (MTs), protein polymers generated from αβ-Tubulin dimers and organized in space and time. Although the last twenty years has resulted in a comprehensive list of proteins essential for organizing mitotic MTs, so called MT associated proteins, or MAPs, and although drugs have been developed that target some of these proteins, the complexity of spindle formation makes it difficult to know how function emerges from component parts. This is especially important given that abnormal mitosis is a hallmark of human pathogenesis, including cancer and neurodegenerative diseases. To understand how faulty or missing gene products contribute to abnormal spindle formation, we must understand the physical properties of individual proteins, both in isolation and together with other proteins, how they are regulated in space and time and how cells activate alternative pathways to compensate for their loss. This requires a multi-scale understanding that encompasses a multi-disciplinary approach. The Drosophila embryo is an excellent model system for studying mitotic spindle formation and, in this thesis, I investigate a conserved MAP, End Binding protein 1 (EB1), which binds autonomously to growing MT plus ends in order to recruit a suite of MT effectors, regulating MT dynamics. Although end binding proteins have been extensively studied, their functions in different stages of mitosis are less understood. In this work, I aimed to investigate the roles of end binding proteins in early Drosophila embryos. To begin with I attempted to generate tools with which to disrupt EB protein function, focusing on interfering antibodies. Next, I used quantitative, comparative mass spectrometry and proteomics to identify proteins that interact with EB1 strongly during metaphase. A simple interpretation of my findings suggests that EB1 interacts with the Dynein/Dynactin complex, HOOK, JVL-SPNF-IK2 complex and PIGS to anchor microtubule ends to the centrosomes and to regulate invagination furrows, mitotic spindle orientation and chromosome segregation. In addition, my results suggest that EB1 interacts with the Rod-ZW10-Zwilch and spindly complex to mediate the interactions with Dynein/Dynactin to silence the spindle assembly checkpoint, leading to the onset of anaphase. Finally, I have identified novel EB1 binding proteins and generated new transgenic flies to investigate their functional relationships with EB1. This study indicates that EB1 interacts with DNApol-ε complex and with Cornetto (CORN). Although their relationships to EB1 and potential function during mitosis remain unknown, their dynamic localisation suggest both may play crucial roles in organising mitotic MTs. My work therefore suggests that EB1 plays crucial roles at the metaphase-anaphase transition, at both kinetochores and the embryo cortex, furthering our understanding of this crucial spindle-regulating protein.