Significance To understand the special morphology of the thumb (digit 1), an important adaptation of the human hand, we studied Hoxa13 -null mice that specifically lack this digit. We show that Hoxa13 mutant limbs specifically lose Hoxd13 expression in the presumptive digit 1 region, correlating with increased Gli3 transcription and Gli3 repressor activity. We also show that Hox13 paralogs regulate Gli3 transcription by negatively modulating the activity of Gli3 enhancers. Our results indicate that mutual antagonism between Gli3 and Hox13 paralogs determines the anterior−posterior asymmetry of the handplate and that Hoxa13 acts hierarchically to initiate formation of a digit 1 territory by attenuating Gli3 transcription and enabling second-phase expansion of 5′Hoxd expression.