Advances in genetic sequencing are now revealing the tremendous diversity of the microbial communities that inhabit or infect living organisms. However, it is difficult to identify the microbes that have the potential to spill over from other species to infect and cause disease in humans. We outline the ecological, epidemiological, and behavioral determinants of pathogen exposure and the within-host biological factors that shape susceptibility to spillover infections. By integrating the insights generated in currently isolated fields, we can quantify barriers to spillover, assess the risk of known pathogens to human health, and identify points of intervention and control.