Abstract XHIM is a congenital immunodeficiency from inadequate CD154 expression on activated CD4 T cells. The only curative therapy is bone marrow transplantation. Recently, lentiviral gene therapy approaches for XHIM have been explored. However, beyond the CD154 transcriptional promoter, the regulatory cis-elements required for cell type-specific, activation dependent, and time-limited expression of CD154 are unclear. We generated a hCD154 BAC transgenic mouse spanning ~143 kb upstream, ~11 kb of the intact hCD154 gene, and ~20 kb of downstream DNA sequence. This BAC contains all known CD154 regulatory elements (promoter, enhancers, 3’ UTR). Confirmation of the intact BAC sequence was determined by DNA sequencing at intervals throughout the BAC. hCD154 transgene expression was detected by flow cytometry. After polyclonal activation of splenocytes, hCD154 is restricted to CD4 T cells (absent on B and CD8 T cells). Activated CD4 T cells co-express mCD154 and hCD154. Like mCD154, hCD154 expression is activation dependent but displays a slightly delayed and prolonged expression. Human CD4 T cells activated for 6 hours and irradiated to prevent division stimulate mCD40 expressing B cells (EdU incorporation), and this is inhibited by anti-hCD154 antibody. By deletion or mutation of known cis-regulatory elements in the hCD154 BAC construct, we will test the minimal requirements for optimal hCD154 expression to stimulate CD40+ cells. This will help design gene therapy vectors for XHIM.