Bacterial artificial chromosome (BAC) modification technology is a powerful method for the identification of enhancer sequences and genetic modifications. Using this method, we have analyzed the Mesp1 and/or Mesp2 enhancers and identified P1-PSME, a PSM-specific enhancer of Mesp1, which contains a T-box binding site similar to the previously identified P2-PSME. Hence, Mesp1 and Mesp2 use different enhancers for their PSM-specific expression. In addition, we find that these two genes also use distinct enhancers for their early mesodermal expression. Based on these results, we generated a PSM-specific Mesp1/Mesp2-null mouse by introducing a BAC clone, from which only early mesodermal Mesp1 expression is possible, into the Mesp1/Mesp2 double knockout (dKO) genetic background. This successfully rescued gastrulation defects due to the lack of the early mesoderm in the dKO mouse and we thereby obtained a PSM-specific Mesp1/Mesp2-null mouse showing a lack of segmented somites.