Janus kinases (Jaks) play an important role in signal transduction via cytokine and growth factor receptors. A targeted inactivation of Jak2 was performed. Jak2^−/− embryos are anemic and die around day 12.5 postcoitum. Primitive erythrocytes are found, but definitive erythropoiesis is absent. Compared to erythropoietin receptor–deficient mice, the phenotype of Jak2 deficiency is more severe. Fetal liver BFU-E and CFU-E colonies are completely absent. However, multilineage hematopoietic stem cells (CD34^low, c-kit^pos) can be found, and B lymphopoiesis appears intact. In contrast to IFNα stimulation, Jak2^−/− cells do not respond to IFNγ. Jak2^−/− embryonic stem cells are competent for LIF signaling. The data provided demonstrate that Jak2 has pivotal functions for signal transduction of a set of cytokine receptors required in definitive erythropoiesis.