In mouse embryos, the Zfhx1 transcription factor genes, Sip1 and δEF1, are expressed in complementary domains in many tissues. Their possible synergism in embryogenesis was investigated by comparing the phenotype of Sip1−/−;δEF1−/− double homozygotes with single homozygous embryos. Unexpectedly, in Sip1−/− embryos δEF1 was ectopically activated, suggesting a negative regulation of δEF1 expression by Sip1. Sip1−/−;δEF1−/− embryos were similar to Sip1−/− embryos in short somite production and developmental arrest around E8.5, but showed more severe defects in dorsal neural tube morphogenesis accompanied by a larger reduction of Sox2 expression, ascribable to the loss of the ectopic δEF1 expression. Sip1+/−;δEF1−/− embryos develop various morphological defects after E10 that were absent in δEF1−/− embryos even in tissues without significant overlap of Sip1 and δEF1 expression, and arrested during mid gestation earlier than δEF1−/− embryos. These findings indicate that complex synergistic interactions occur between Zfhx1 transcription factor genes during mouse embryogenesis. Developmental Dynamics 235:1941–1952, 2006. © 2006 Wiley-Liss, Inc.