In order to understand the controls of gene expression in hematopoietic stem cells (HSC), we have studied the regulation of human CD34, a cell surface glycoprotein used in isolation of HSC. We previously reported that regions 10 to 18 kb 5′ upstream of the transcription start site (TSS) and/or 17 to 26 kb 3′ downstream of the transcription termination site (TTS) of the human CD34 gene contain critical elements for expression in vivo. Mice transgenic for a human CD34 PAC clone (PAC54A19) that includes 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequences expressed human CD34 antigen in the majority of HSC. Because these transgenic mice also demonstrated downregulation of human CD34 gene expression with maturation of hematopoietic cells, we concluded that these critical cis-elements are necessary not only for human CD34 expression in immature cells, but also for downregulation in mature cells. To further delineate the distal cis-elements which regulate the human CD34 gene, we generated two deletion constructs which removed sequences either upstream from −12.8 kb (PmeI deletion) or downstream of +18.2 kb (FseI deletion) of the human CD34 gene. We used a murine myeloblastic cell line, 416B, to test these constructs. We first generated stable clones of 416B cells with PAC54A19; these stable clones expressed human CD34 RNA and protein. In contrast, neither PmeI nor FseI deletion constructs conferred human CD34 mRNA or protein expression in these 416B stable cell lines. Therefore, we conclude that both 5′ upstream and 3′ downstream distal cis-elements are necessary for proper human CD34 expression in cell lines. We also performed DNaseI hypersensitivity assays to localize open chromatin structures in the human CD34 gene in several human myeloblastic cell lines. We detected one DNaseI hypersensitive site (DHS) −12.5 kb upstream of the TSS and several DHS in the region located between +17 kb to +26 kb 3′ downstream of the human CD34 TTS in the human myeloblastic CD34+ KG1a cell line. In addition, we performed similarity searches of the −18 kb to −10 kb human CD34 5′ flanking sequence and +17 kb to +26 kb human CD34 3′ flanking sequence, comparing with the murine CD34 genomic sequence. We identified one conserved region in the −11.5 kb 5′ upstream region of the TSS and one highly conserved region located +19 kb 3′ downstream region of the TTS. To further localize distal cis-elements, we generated several deletion constructs in the parental PAC54A19 PAC clone using the ET cloning system, which facilitates manipulation of PACs by homologous recombination in bacteria. Using this method, we showed that deletion of a 600 bp region located 15 kb 5′ of the TSS or a 1000 bp sequence located 19 kb downstream of the human CD34 gene led to loss of human CD34 expression in 416B stable lines. Our current efforts are focused on identifying the transcription factors that bind to these regions to regulate the expression of human CD34 in stem cells.