Natalizumab is a highly effective immunomodulator in the treatment of multiple sclerosis (MS). Treatment with natalizumab has been associated with progressive multifocal leukoencephalopathy (PML), an infection of the central nervous system (CNS) caused by a pathogenic form of the normally benign JC virus (JCV). We searched PubMed and used current data from the natalizumab global safety database to assess risk factors and quantify the risk of PML. Natalizumab treatment duration and prior use of immunosuppressive therapies are established risk factors for development of PML in natalizumab-treated patients. With the development of a reliable and validated assay for detection of antibodies in patients with MS directed against JCV, it is now possible to identify persons who are carriers of JCV. The availability of this assay provides an additional option for risk stratification of PML in patients using or considering natalizumab therapy. Recommendations for clinical management of patients with MS and use of natalizumab are provided based on the presence of these three risk factors. The identification of risk factors that increase the likelihood of PML in natalizumab-treated patients can facilitate benefit-risk discussions between health care professionals and patients. Continued research and data collection will further develop our understanding of PML and the mechanisms by which these risk factors contribute to its development.