Mice carrying a conditional prothrombin knockout allele (fII^lox) were established to develop an experimental setting for exploring the importance of thrombin in the maintenance of vascular integrity, the inflammatory response, and disease processes in adult animals. In the absence of Cre-mediated recombination, homozygous fII^lox/lox mice or compound heterozygous mice carrying one fII^lox allele and one constitutive-null allele were viable. Young adults exhibited neither spontaneous bleeding events nor diminished reproductive success. However, the induction of Cre recombinase in fII^lox mice using the poly I:C-inducible Mx1-Cre system resulted in the rapid and near-complete recombination of the fII^lox allele within the liver, the loss of circulating prothrombin, and profound derangements in coagulation function. Consistent with the notion that thrombin regulates coagulation and inflammatory pathways, an additional early consequence of reducing prothrombin was impaired antimicrobial function in mice challenged with Staphylococcus aureus peritonitis. However, life expectancy in unchallenged adults genetically depleted of prothrombin was very short (∼5-7 days). The loss of viability was associated with the development of severe hemorrhagic events within multiple tissues, particularly in the heart and brain. Unlike the constitutive loss of either clotting or platelet function alone, the conditional loss of prothrombin is uniformly not compatible with maintenance of hemostasis or long-term survival.