AbstractTranscriptional control has been identified as a key mechanism regulating the formation and subsequent behavior of hematopoietic stem cells. We have used a comparative genomics approach to identify transcriptional regulatory elements of the LMO2 gene, a transcriptional cofactor originally identified through its involvement in T-cell leukemia and subsequently shown to be critical for normal hematopoietic and endothelial development. Of the 2 previously characterized LMO2 promoters, the second (proximal) promoter was highly conserved in vertebrates ranging from mammals to fish. Real-time reverse transcriptase–polymerase chain reaction (RT-PCR) expression analysis identified this promoter as the predominant source of transcription in hematopoietic tissue. Transient and stable transfections indicated that the proximal promoter was active in hematopoietic progenitor and endothelial cell lines and this activity was shown to depend on 3 conserved Ets sites that were bound in vivo by E74-like factor 1 (Elf1), Friend leukemia integration 1 (Fli1), and erythroblastosis virus oncogene homolog E twenty-six–1 (Ets1). Finally, transgenic analysis demonstrated that the LMO2 proximal promoter is sufficient for expression in endothelial cells in vivo. No hematopoietic expression was observed, indicating that additional enhancers are required to mediate transcription from the proximal promoter in hematopoietic cells. Together, these results suggest that the conserved proximal promoter is central to LMO2 transcription in hematopoietic and endothelial cells, where it is regulated by Ets factors.