Increasing direct and indirect evidence suggests that erythropoietin (Epo) promotes both erythropoiesis and megakaryocytopoiesis. Here we report that, in mice infected with a recombinant spleen focus-forming retrovirus (SFFV) expressing an oncogenic erythropoietin receptor (EpoR), there was an increase in platelet count preceding the ensuing erythrocytosis. Concurrently, there was a substantial increase in splenic megakaryocytes. Culture of the bone marrow and spleen cells from infected mice showed enhanced numbers of multipotent megakaryocytic progenitors. DNA polymerase chain reaction analysis of individual megakaryocyte-containing colonies showed recombinant SFFV (SFFVcEpoR) proviral integration. Immunofluorescence of spleen sections showed overexpression of EpoR protein in the megakaryocytes. Mice infected with a strain of SFFV also developed splenic megakaryocytosis without activating overexpression of the EpoR in megakaryocytes. This in vivo system shows that a relationship between erythropoiesis and thrombopoiesis can exist at the level of the Epo-EpoR signaling pathway. Also, SFFV-based vectors may be excellent vehicles for the introduction of genes into multipotent, hematopoietic progenitors, in vitro.