Hematopoietic defects in HOXA9^–/– mice demonstrate a key role for this homeoprotein in blood cell development. Conversely, enforced HOXA9 expression is leukemogenic in mice, and HOXA9 is frequently activated in human acute myeloid leukemia (AML). Although HOXA9 is thought to function as a transcription factor, few downstream targets have been identified. We searched for early HOXA9 target genes by using a transient overexpression strategy in 3 hematopoietic cell lines (2 myeloid, 1 lymphoid). cDNA microarray analyses identified 220 genes whose expression was modulated at least 2-fold. Expression signatures in myeloid and lymphoid cells demonstrated that HOXA9 functions as both an activator and repressor of a variety of genes in cell-specific patterns suggesting that the transcriptional effects of HOXA9 are largely dependent on the cell context. Transient transcription assays and target gene expression patterns in HOXA9^–/– marrow cells imply that we have identified direct physiologic targets. Many target genes are expressed in CD34⁺ stem cells or are members of gene families involved in proliferation or myeloid differentiation. Expression of 14 HOXA9 target genes correlated with high-level HOXA9 expression in primary AML. These data suggest that many genes identified in this survey may mediate the biologic effects of HOXA9 in normal and leukemic hematopoiesis.