Chemical genomics involves generating large collections of small molecules and using them to modulate cellular states. Despite recent progress in the systematic synthesis of structurally diverse compounds, their use in screens of cellular circuitry is still an ad hoc process 1-4 . Here, we outline a general, efficient approach called gene expression-based highthroughput screening (GE-HTS) in which a gene expression signature is used as a surrogate for cellular states, and we describe its application in a particular setting: the identification of compounds that induce the differentiation of acute myeloid leukemia cells. In screening 1,739 compounds, we identified 8 that reliably induced the differentiation signature and, furthermore, yielded functional evidence of bona fide differentiation. The results indicate that GE-HTS may be a powerful, general approach for chemical screening.To prove the feasibility of GE-HTS, we applied the method to the identification of compounds capable of inducing terminal differentiation of acute myelogenous leukemia (AML) cells. The plausibility of differentiation induction in leukemia is suggested by a rare subtype of AML known as acute promyelocytic leukemia (APL) in which treatment with all-trans retinoic acid (ATRA) results in clinical remissions through modulation of a mutated retinoic acid receptor alpha 5,6 . Unfortunately, ATRA has no clinical efficacy in other subtypes of AML 7 . But the fact that all forms of AML show a block of differentiation suggests that differentiation therapy might be possible, provided that the right compounds could be identified. The mechanism underlying such blocked differentiation is unknown in most cases; thus, it is not possible to carry out small-molecule screens against a validated target protein. Instead, a cell-based screening approach is needed. The usual approach to assaying the myeloid differentiation phenotype, however, involves a combination of visual inspection of nuclear morphology and biochemical tests, such as nitroblue tetrazolium (NBT) reduction 8 , neither of which readily lend themselves to a highthroughput screening platform. We therefore sought to use GE-HTS to identify compounds capable of inducing the differentiation program in AML without first knowing the crucial targets of this process.The first step in the GE-HTS procedure is defining the gene expression signatures of the biological states of interest. To accomplish this, we carried out oligonucleotide microarray-based gene expression profiling of pretreatment bone marrow samples derived from individuals affected with AML, as well as of fully differentiated peripheral blood neutrophils and monocytes derived from unaffected donors (gene expression data are available in Supplementary Table 1a,b online). We thereby identified genes correlated with the neutrophil-versus-AML or monocyte-versus-AML distinctions (Fig. 1). We selected from these differentiation-correlated genes a handful of marker genes that represented the diversity of myeloid differentiation yet could be easil...