To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.