To elucidate the physiological significance of MEK5 in vivo, we have examined the effect of mek5 gene elimination in mice. Heterozygous mice appear to be healthy and were fertile. However, mek5-/- embryos die at approximately embryonic day 10.5 (E10.5). The phenotype of the mek5 -/- embryos includes abnormal cardiac development as well as a marked decrease in proliferation and an increase in apoptosis in the heart, head, and dorsal regions of the mutant embryos. The absence of MEK5 does not affect cell cycle progression but sensitizes mouse embryonic fibroblasts (MEFs) to the ability of sorbitol to enhance caspase 3 activity. Further studies with mek5-/- MEFs indicate that MEK5 is required for mediating extracellular signal-regulated kinase 5 (ERK5) activation and for the regulation of the transcriptional activity of myocyte enhancer factor 2. Overall, this is the first study to rigorously establish the role of MEK5 in vivo as an activator of ERK5 and as an essential regulator of cell survival that is required for normal embryonic development.