Protein kinase CK2 is a ubiquitous protein kinase implicated in proliferation and cell survival. Its regulatory beta subunit, CK2beta, which is encoded by a single gene in mammals, has been suspected of regulating other protein kinases. In this work, we show that knockout of the CK2beta gene in mice leads to postimplantation lethality. Mutant embryos were reduced in size at embryonic day 6.5 (E6.5). They did not exhibit signs of apoptosis but did show reduced cell proliferation. Mutant embryos were resorbed at E7.5. In vitro, CK2beta(-/-) morula development stopped after the blastocyst stage. Attempts to generate homozygous embryonic stem (ES) cells failed. By using a conditional knockout approach, we show that lack of CK2beta is deleterious for mouse ES cells and primary embryonic fibroblasts. This is in contrast to what occurs with yeast cells, which can survive without functional CK2beta. Thus, our study demonstrates that in mammals, CK2beta is essential for viability at the cellular level, possibly because it acquired new functions during evolution.