Mammalian sterile 20-like kinase 1 (Mst1) is a ubiquitously expressed serine/threonine kinase belonging to the family of Sterile 20-like kinases. MST1 has been inferred to play important roles in apoptosis and in the inhibition of proliferation in mammalian cells. Here, we describe the genetic characterization of Mst1-deficient mice produced by two distinct gene-trap insertions. Animals generated from clone RRT293 exhibit transmission ratio distortion favoring the mutated allele which is amplified with each generation. Inexplicably, while the mutated allele is favored for transmission, its homozygosity is embryonic lethal. By contrast, animals generated from the second Mst1 gene-trap clone, AJ0315, do not show any gross abnormalities. We find that the discrepancy in phenotype is most likely attributable to a second insertion in the RRT293 clone. Thus, a mutation in Mst1 alone does not affect survival. Our results set the stage for identification of the lethal second-site mutation that is paradoxically favored for transmission.