During cellular differentiation and development, it is recognized that many complex molecular mechanisms as well as precise patterns of differentially expressed genes occur in directing precursor cells toward a given lineage. Using microarray-based technology, we examined gene expression across the course of 3T3-L1 adipocyte differentiation. Total cellular RNA was isolated at times 0, 2, 8, 16, 24, 48, and 96 h following treatment with either standard hormonal inducers of differentiation; insulin, dexamethasone, isobutylmethylxanthine (IDX), or IDX plus trichostatin A (TsA), a histone deacetylase inhibitor and potent adipogenic inhibitor. cRNA was synthesized from cellular RNA and hybridized to high density Affymetrix MG_U74Av2 microarray gene chips containing 12,488 cDNA/Expressed Sequence Tags (ESTs) probe sets. From the IDX-only treated cells, all probe sets that were either unchanged or differentially expressed less than 2-fold throughout differentiation with respect to time 0 preadipocytes were excluded from further analyses. This selection resulted in a net of 1686 transcripts, 859 were increased in expression, and 827 were decreased in expression at least 2-fold across differentiation. To focus in on genes that were more specific to differentiation, the same analysis was performed on IDX plus TsA-treated non-differentiating cells and all probe sets from the IDX-only group that exhibited similar expression profiles in the non-differentiating TsA-treated group were excluded leaving a total of 1016 transcripts that were regulated only under differentiating conditions. Six hundred and thirty-six of these transcripts were elevated at least 2-fold and 380 exhibited a decrease in expression relative to time 0 preadipocytes. This group of genes was further analyzed using hierarchical clustering and self-organizing maps and resulted in the identification of numerous genes not previously known to be regulated during adipocyte differentiation. Many of these genes may well represent novel adipogenic mediators and markers of adipogenesis.