The role of the cytosolic domain of tissue factor (TT) in signal transduction and gene regulation was studied in mice with a targeted deletion of the 18 carboxyterminal intracellular amino acids. This deletion was introduced in exon 6 along with a floxed neo(R) selection cassette in intron 5 using homologous recombination in embryonic stem cells. Removal of the floxed neo(R) cassette by in vivo Cre-mediated loxP recombination yielded TF+/delta CT and TFdelta CT/delta CT mice. In contrast to TF-/- mice, TF+/delta CT and TFdelta CT/delta CT mice displayed normal embryonic development, survival, fertility, and blood coagulation. Factor VIIa or factor Xa stimulation produced similar p44/42 MAPK activation in TF+/+ and TFdelta CT/delta CT fibroblasts. These data, based on expression of a TFdelta CT molecule from the endogenous TF locus, provide conclusive proof that the cytosolic domain of TF is not essential for signal transduction in embryogenesis and in physiological postnatal processes.