Ebola virus (EBOV) is a lipid-enveloped virus with a high fatality rate. With only seven genes in the Ebola genome, how is a deadly infection scheme achieved? A critical component of the virus is VP40, the matrix protein. Not only is this protein the most abundantly expressed of the virus, it is also a protein that can multitask in structure and biological function. One of VP40's roles is to bind the host plasma membrane (PM) and bridge the viral nucleocapsid upon exiting the host cell. Surprisingly, VP40 is sufficient to form the virus particle without any other EBOV components. Particles formed by VP40 without the other viral components are called virus like particles (VLPs). VLPs are non-infections and thus an excellent tool to study VP40 function in live cells. VLPs are formed through two VP40 actions: 1. VP40 host lipid binding and 2. VP40 oligomer formation. Both events are believed to occur at the PM. VP40 lipid binding and VP40 oligomerization are closely related as lipid binding promotes and stabilizes the formation of oligomers. VP40 lipid binding and oligomerization are both required for VLP egress and thus EBOV to be infectious. The work presented in this thesis aims to understand the mechanism of VP40 membrane binding and VP40 oligomer formation. A variety of biochemical techniques to visualize, detect, and quantify changes in VP40 lipid binding and oligomer formation were used in live cells and in vitro to better understand how these processes occur. Previous work in the lab found that VP40 required phosphatidylserine (PS) for PM association and VLP budding. PS was also required for VP40 hexamer formation at the PM. In this thesis, we found that in addition to PS, VP40 binds phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) with high affinity and selectivity at the PM. This interaction seems to form and stabilize larger oligomers built from hexamer subunits. PI(4,5)P2 is also required for efficient VLP formation and egress, suggesting the stability of large VP40 structures is necessary for efficient viral egress. We now understand that the interaction between VP40 and PI(4,5)P2 occurs through C-terminal domain lysine residues. Since VP40 is necessary for the structural integrity of the virus, our results reveal the molecular underpinnings of how this process might occur. Thus, the VP40-PI(4,5)P2 binding site is an excellent candidate for site-directed inhibition. Remarkably, VP40 membrane sensing and binding extends beyond the lipid headgroup. VP40 binding is significantly influenced by the membrane packing density (i.e., fluidity). Ordered membranes containing PS or PI(4,5)P2 illicit robust VP40 binding while disordered membranes containing PS or PI(4,5)P2 exhibit no significant binding. While the relationship between VP40 and membrane fluidity is still under investigation, it holds potential for a novel therapeutic approach against EBOV and potentially other filoviruses. Overall, the work presented in this thesis establishes novel insights into VP40 lipid binding and the mechanism by which EBOV forms from the PM of the host cell.