Epstein-Barr Virus (EBV) is a B-lymphotropic herpesvirus which the majority of adult human population is latently-infected with. Various immunological and molecular in vitro studies have been facilitated by the use of EBV’s ability to infect and transform B cells to immortalized polyclonal B cell lines. Many of these studies use freshly isolated cord-blood mononuclear cells (CBMC). Some experiments may, however, require EBV infection of samples that have been prospectively collected and cryo-preserved. Here we share a protocol that we used to successfully infect B cells from cryo-preserved CBMCs and peripheral-blood mononuclear cells (PBMC) (Sohlberg et al., 2013; Saghafian-Hedengren et al., 2013).