AbstractTo gain insight into the coordination of gene expression profiles during forelimb and hindlimb differentiation, a transcriptome analysis of mouse embryonic autopod tissues was performed using Affymetrix Murine Gene Chips (MOE‐430). Forty‐four transcripts with expression differences higher than 2‐fold (T test, P ≤0.05) were detected between forelimb and hindlimb tissues including 38 new transcripts such as Rdh10, Frzb, Tbx18, and Hip that exhibit differential limb expression. A comparison of gene expression profiles in the forelimb, hindlimb, and brain revealed 24 limb‐signature genes whose expression was significantly enriched in limb autopod versus brain tissue (fold change >2, P ≤ 0.05). Interestingly, the genes exhibiting enrichment in the developing autopod also segregated into significant fore‐ and hindlimb‐specific clusters (P ≤ 0.05) suggesting that by E 12.5, unique gene combinations are being used during the differentiation of each autopod type. Developmental Dynamics 234:74–89, 2005. © 2005 Wiley‐Liss, Inc.