Mammalian GATA2 gene encodes a dual zinc finger transcription factor, which is essential for hematopoietic stem cell (HSC) generation in the aorta, gonad, mesonephros (AGM) region, HSC self-renewal, and specification of progenitor cell fates. Previously, we demonstrated that Gata2 expression in AGM is controlled by its intronic +9.5 enhancer. Gata2 +9.5 deficiency removes the E-box motif and the GATA site and depletes fetal liver HSCs. However, whether this enhancer has an essential role in regulating adult hematopoiesis has not been established. Here, we evaluate Gata2 +9.5 enhancer function in adult hematopoiesis. +9.5^+/− bone marrow cells displayed reduced T cell reconstitution in a competitive transplant assay. Donor-derived analysis demonstrated a previously unrecognized function of the +9.5 enhancer in T cell development at the lymphoid-primed multipotent progenitor stage. Moreover, +9.5^+/− adult HSCs displayed increased apoptosis and reduced long-term self-renewal capability in comparison with wild-type (WT) HSCs. These phenotypes were more moderate than those of Gata2^+/− HSCs. Consistent with the phenotypic characterization, Gata2 expression in +9.5^+/− LSKs was moderately higher than that in Gata2^+/− LSKs, but lower than that in WT LSKs. Our data suggest that +9.5 deficiency compromises, without completely abrogating, Gata2 expression in adult HSCs.