Self-renewal and multipotency are essential functions of hematopoietic stem cells (HSCs). To maintain homeostatic hematopoiesis, functionally uniform HSCs have been thought to be an ideal cell-of-origin. Recent technological advances in the field have allowed us to analyze HSCs with single cell resolution and implicate that functional heterogeneity may exist even within the highly purified HSC compartment. However, due in part to the technical limitations of analyzing extremely rare populations and our incomplete understanding of HSC biology, neither the biological meaning of why heterogeneity exists nor the precise mechanism of how heterogeneity is determined within the HSC compartment is entirely known. Here we show the first evidence that self-renewal capacity varies with the degree of replication stress dose and results in heterogeneity within the HSC compartment. Using the Hoxb5-reporter mouse line which enables us to distinguish between long-term (LT)-HSCs and short-term (ST)-HSCs, we have found that ST-HSCs quickly lose self-renewal capacity under high stress environments but can maintain self-renewal under low stress environments for long periods of time. Critically, exogeneous Hoxb5 expression confers protection against loss of self-renewal to Hoxb5-negative HSCs and can partially alter the cell fate of ST-HSCs to that of LT-HSCs. Our results demonstrate that Hoxb5 imparts functional heterogeneity in the HSC compartment by regulating self-renewal capacity. Additionally, Hoxb5-positive HSCs may exist as fail-safe system to protect from the exhaustion of HSCs throughout an organism's lifespan. (C) 2021 The Authors. Published by Elsevier Inc.