The p53 protein can inhibit cell cycling or induce apoptosis, and is thus a critical regulator of tumorigenesis. This protein is negatively regulated by a physical interaction with MDM2, an E3 ubiquitin ligase. This interaction is critical for cell viability; loss of Mdm2 causes cell death in vitro and in vivo in a p53-dependent manner. The recently discovered MDM2-related protein MDM4 (also known as MDMX) has some of the same properties as MDM2. MDM4 binds and inhibits p53 transcriptional activity in vitro. Unlike MDM2, however, MDM4 does not cause nuclear export or degradation of p53 (refs. 9,10). To study MDM4 function in vivo, we deleted Mdm4 in mice. Mdm4-null mice died at 7.5-8.5 dpc, owing to loss of cell proliferation and not induction of apoptosis. To assess the importance of p53 in the death of Mdm4-/- embryos, we crossed in the Trp53-null allele. The loss of Trp53 completely rescued the Mdm4-/- embryonic lethality. Thus, MDM2 and MDM4 are nonoverlapping critical regulators of p53 in vivo. These data define a new pathway of p53 regulation and raise the possibility that increased MDM4 levels and the resulting inactivation of p53 contribute to the development of human tumors.