Staphylococcus aureus is defined by its ability to agglutinate during exposure to human blood plasma. Although agglutination has long correlated with disease severity, the function of agglutination during infection remains unclear. Increasing evidence suggests the mechanisms of agglutination are highly complex and poorly understood. The goal of this dissertation was to characterize the mechanisms required for S. aureus agglutination in vitro and determine how these factors contribute to pathogenesis. Chapter II focuses on the development of two in vitro agglutination assays, which allow the process to be measured quantitatively. Through these assays, we confirmed the major factors contributing to agglutination are human fibrinogen and the bacterial surface protein, ClfA. Productive interactions between these two factors are required for agglutination to proceed. Surprisingly, we also identified a novel regulatory system that significantly contributed to agglutination. Inactivation of the ArlRS two-component system (TCS) prevents agglutination in both of the developed assays. Studies in Chapter III focused on characterizing the mechanism by which ArlRS inhibits agglutination. To examine regulation, quantitative PCR identified the major output of the ArlRS system as the gene ebh. Surprisingly, transcript levels of known extracellular matrix (ECM) binding proteins did not change. Characterization of ebh indicated that overexpression in an arlRS mutant is the major factor responsible for preventing agglutination. Deletion of ebh restores the ability of the arlRS mutant to agglutinate in both gravity and flow-based agglutination assays. Fluorescence microscopy of clumps indicates wildtype cells bind and incorporate fluorescently labeled human fibrinogen (Fg) displaying co-localization with the clumps. Surprisingly, arlRS mutants also bound human Fg, but these interactions were not productive for clumping, suggesting successful agglutination is more complex than binding ECM proteins. These studies indicate that ArlRS regulates agglutination through a unique mechanism that depends on the surface protein Ebh. Studies in Chapter IV were performed to determine the role ArlRS played in pathogenesis. A rabbit model of infective endocarditis and sepsis was employed to assess ArlRS virulence because this model has been shown to require agglutination for disease progression. Mutants in arlRS displayed reduced virulence in the rabbit model of infective endocarditis, which correlated with the mutant's inability to form a vegetation of the heart valve. These studies provide further insight into the importance of S. aureus agglutination during infection and define a mechanism of regulation through a novel surface protein.