The relationship between gene network structure and expression variation among individuals and species: Variation among individuals is a prerequisite of evolution by natural selection. As such, identifying the origins of variation is a fundamental goal of biology. We investigated the link between gene interactions and variation in gene expression among individuals and species, using the mammalian limb as a model system. We first built interaction networks for key genes regulating early (outgrowth; E9.5-11) and late (expansion and elongation; E11-13) limb development in mouse. This resulted in an Early (ESN) and Late (LSN) Stage Network. Computational perturbations of these networks suggest that the ESN is more robust. We then quantified levels of the same key genes among mouse individuals, and found that they vary less at earlier limb stages and that variation in gene expression is heritable. Finally, we quantified variation in gene expression levels among four mammals with divergent limbs (bat, opossum, mouse and pig), and found that levels vary less among species at earlier limb stages. We also found that variation in gene expression levels among individuals and species are correlated for earlier and later limb development. In conclusion, results are consistent with the robustness of the ESN buffering among-individual variation in gene expression levels early in mammalian limb development, and constraining the evolution of early limb development among mammalian species. Transcriptomic insights into the genetic basis of mammalian limb diversity: From bat wings to whale flippers, limb diversification has been crucial to the evolutionary success of mammals. We performed the first transcriptome-wide study of limb development in multiple species to explore the hypothesis that mammalian limb diversification has proceeded through the differential expression of conserved shared genes, rather than by major changes to limb patterning. Specifically, we investigated the manner in which the expression of shared genes has evolved within and among mammalian species. We assembled and compared transcriptomes of bat, mouse, opossum, and pig fore- and hind limbs at the ridge, bud, and paddle stages of development. Results suggest that gene expression patterns exhibit larger variation among species during later than earlier stages of limb development, while within species results are more mixed. Consistent with the former, results also suggest that genes expressed at later developmental stages tend to have a younger evolutionary age than genes expressed at earlier stages. A suite of key limb-patterning genes was identified as being differentially expressed among the homologous limbs of all species. However, only a small subset of shared genes is differentially expressed in the fore- and hind limbs of all examined species. Similarly, a small subset of shared genes is differentially expressed within the fore- and hind limb of a single species and among the forelimbs of different species. Taken together, results of this study do not support the existence of a phylotypic period of limb development ending at chondrogenesis, but do support the hypothesis that the hierarchical nature of development translates into increasing variation among species as development progresses. Overall design: Bat, mouse, opossum, and pig mRNA profiles at early and late developmental stages on each species fore and hind-limbs. Various replicates of each library were generated by single-end sequencing using Illumina HiSeq 2500. Please note that the De novo transcriptome assembly for bat (Trinity.fasta) was generated from pooled RNA-seq data of fore and hind-limbs at various embryonic developmental stages; Beginning stage (Wanek stage 2: 3 FL and 3 HL samples), early-stage (Wanek stage 3/4: 2 FL and 2 HL samples), and late_stage (Wanke stage 6: 2 FL and 2 HL samples). Gene expression libraries (RNA-seq) were created at ridge, bud, and paddle developmental stages among fore and hind-limbs of bat, mouse, opossum, and pig. Various replicates of each condition were generated by single-end sequencing using Illumina HiSeq 2500. The ridge, bud, and paddle stages were defined based on morphology and correspond to the following embryonic developmental stages: W2, W3, and W6, respectively, on mouse; 13, 14, and 15, respectively, on bat; 27/30 (fore/hindlimb), 31, and 32, respectively, on opossum; and 20, 22, and 26, respectively, on pig.