Btg1 and Btg2 encode highly homologous proteins that are broadly expressed in different cell lineages, and have been implicated in different types of cancer. Btg1 and Btg2 have been shown to modulate the function of different transcriptional regulators, including Hox and Smad transcription factors. In this study, we examined the in vivo role of the mouse Btg1 and Btg2 genes in specifying the regional identity of the axial skeleton. Therefore, we examined the phenotype of Btg1 and Btg2 single knockout mice, as well as novel generated Btg1-/-;Btg2-/- double knockout mice, which were viable, but displayed a non-mendelian inheritance and smaller litter size. We observed both unique and overlapping phenotypes reminiscent of homeotic transformation along the anterior-posterior axis in the single and combined Btg1 and Btg2 knockout animals. Both Btg1-/- and Btg2-/- mice displayed partial posterior transformation of the seventh cervical vertebra, which was more pronounced in Btg1-/-;Btg2-/- mice, demonstrating that Btg1 and Btg2 act in synergy. Loss of Btg2, but not Btg1, was sufficient for complete posterior transformation of the thirteenth thoracic vertebra to the first lumbar vertebra. Moreover, Btg2-/- animals displayed complete posterior transformation of the sixth lumbar vertebra to the first sacral vertebra, which was only partially present at a low frequency in Btg1-/- mice. The Btg1-/-;Btg2-/- animals showed an even stronger phenotype, with L5 to S1 transformation. Together, these data show that both Btg1 and Btg2 are required for normal vertebral patterning of the axial skeleton, but each gene contributes differently in specifying the identity along the anterior-posterior axis of the skeleton.