Kdm6b (JMJD3) is one of two known epigenetic modifiers responsible for the removal of the repressive histone mark, histone-3 lysine-27 trimethylation (H3K27me3), and has been shown to play a role in development, differentiation, and inflammatory stress response. Unlike the other H3K27me3 demethylase, UTX (KDM6A), which is frequently mutated in hematopoietic malignancies, Kdm6b is upregulated in a myriad of blood disorders. This suggests that it may have important functions in the pathogenesis of hematopoietic cancers. Here, we examined the role of Kdm6b in hematopoietic stem cell (HSC) fate decisions under normal and malignant conditions to evaluate its potential as a therapeutic target. Loss of Kdm6b leads to a significant reduction in phenotypic and functional HSCs in adult mice, which increases with increased age. Loss of Kdm6b results in the inability to maintain the HSC population post-transplantation in a dose-dependent manner. In addition, Kdm6b is necessary for HSC self-renewal in response to inflammatory, genotoxic and oncogenic stress. Kdm6b HSCs have a stress response gene expression signature which overlaps significantly with immediate early response genes, genes associated with aged HSCs and genes involved in quiescence of HSCs. When stimulated with an inflammatory or proliferative agent, Kdm6b--deficient HSCs are not able to efficiently resolve gene expression programs, leading to delayed cell cycle entry and a self-renewal block, forcing them to differentiate once they commit to divide. Thus, Kdm6b is necessary for self-renewal of normal and leukemic stem cells, and Kdm6b inhibition combined with proliferative agents may force differentiation and eventual depletion of leukemic stem cells in patients.