AbstractThe ubiquitination status of RIPK1 is considered to be critical for cell fate determination. However, the in vivo role for RIPK1 ubiquitination remains undefined. Here we show that mice expressing RIPK1K376R which is defective in RIPK1 ubiquitination die during embryogenesis. This lethality is fully rescued by concomitant deletion of Fadd and Ripk3 or Mlkl. Mechanistically, cells expressing RIPK1K376R are more susceptible to TNF-α induced apoptosis and necroptosis with more complex II formation and increased RIPK1 activation, which is consistent with the observation that Ripk1K376R/K376R lethality is effectively prevented by treatment of RIPK1 kinase inhibitor and is rescued by deletion of Tnfr1. However, Tnfr1−/−Ripk1K376R/K376R mice display systemic inflammation and die within 2 weeks. Significantly, this lethal inflammation is rescued by deletion of Ripk3. Taken together, these findings reveal a critical role of Lys376-mediated ubiquitination of RIPK1 in suppressing RIPK1 kinase activity–dependent lethal pathways during embryogenesis and RIPK3-dependent inflammation postnatally.