EBV, a human herpesvirus, is commonly acquired during childhood and persists latently in B cells. EBV seropositivity has been connected to immunomodulatory effects such as altered T and NK cell functional responses as well as protection against early IgE sensitization; however, owing to the asymptomatic presentation during childhood little is known regarding the infection process in children of different ages. In this study, we used mononuclear cells from cord blood and from 2- and 5-y-old EBV-naive children for in vitro EBV infection. We show that the degree of EBV-induced B cell activation and expansion differs between age groups and in particular in relationship to IFN-γ production capacity. EBV infection induced redistribution between B cell subsets with enrichment of IgD(+)CD27(+) cells (commonly referred to as non-switched memory) in infected cord blood cell cultures, and of IgD(-)CD27(+) cells (switched memory) in cell cultures from older children. We also related results to serostatus to CMV, a persistent herpesvirus that can affect differentiation status of T and NK cells. As compared with CMV(-) children, the EBV-induced enrichment of IgD(-)CD27(+) B cells was significantly reduced in infected cell cultures from CMV(+) children. This effect was associated with high levels of IFN-γ and frequencies of highly mature CD8(+)CD57(+) T cells in CMV(+) children. Our results demonstrate that both a child's age and serostatus to CMV will have an impact on EBV-induced B cell activation and expansion, and they point to the ability of viruses with immunomodulatory functions, such as CMV, to affect immune responses within the host system.