The elements regulating gene expression in hematopoietic stem cells are still poorly understood. We previously reported that a 141-kilobase (kb) human CD34 transgene confers properly regulated human CD34 expression in transgenic mice. A construct with only the human CD34 promoter and 3′ enhancer region is not sufficient, suggesting that critical distal elements are necessary for expression of the human CD34 gene. To further localize such elements, we analyzed deletion constructs of the human CD34 gene and evaluated their function in transgenic mice. Constructs harboring as little as 18 kb of 5′ and 26 kb of 3′ human CD34 flanking sequence conferred human expression in tissues of transgenic mice with a pattern similar to that of the 141-kb human transgene. In contrast, a construct harboring 10 kb of 5′ and 17 kb of 3′ human CD34 flanking sequence gave no expression. These data demonstrate that regions between 10 to 18 kb upstream and/or 17 to 26 kb downstream of the human CD34 gene contain critical elements for human CD34 expression in vivo. Further functional analysis of these regions in transgenic mice will be crucial for understanding CD34 gene expression in hematopoietic stem and progenitor cells.