Parvovirus B19 (B19V) infection is highly restricted to human erythroid progenitor cells. Although previous studies have led to the theory that the basis of this tropism is receptor expression, this has been questioned by more recent observation. In the study reported here, we have investigated the basis of this tropism, and a potential role of erythropoietin (Epo) signaling, in erythroid progenitor cells (EPCs) expanded ex vivo from CD34(+) hematopoietic cells in the absence of Epo (CD36(+)/Epo(-) EPCs). We show, first, that CD36(+)/Epo(-) EPCs do not support B19V replication, in spite of B19V entry, but Epo exposure either prior to infection or after virus entry enabled active B19V replication. Second, when Janus kinase 2 (Jak2) phosphorylation was inhibited using the inhibitor AG490, phosphorylation of the Epo receptor (EpoR) was also inhibited, and B19V replication in ex vivo-expanded erythroid progenitor cells exposed to Epo (CD36(+)/Epo(+) EPCs) was abolished. Third, expression of constitutively active EpoR in CD36(+)/Epo(-) EPCs led to efficient B19V replication. Finally, B19V replication in CD36(+)/Epo(+) EPCs required Epo, and the replication response was dose dependent. Our findings demonstrate that EpoR signaling is absolutely required for B19V replication in ex vivo-expanded erythroid progenitor cells after initial virus entry and at least partly accounts for the remarkable tropism of B19V infection for human erythroid progenitors.