Haematopoietic stem cells (HSCs) reside in specialized microenvironments, also referred to as niches, and it has been widely believed that HSC numbers are determined by the niche size alone 1–5 . However, the vast excess of the number of niche cells over that of HSCs raises questions about this model. We initially established a mathematical model of niche availability and occupancy, which predicted that HSC numbers are restricted at both systemic and local levels. To address this question experimentally, we developed a femoral bone transplantation system, enabling us to increase the number of available HSC niches. We found that the addition of niches does not alter total HSC numbers in the body, regardless of whether the endogenous (host) niche is intact or defective, suggesting that HSC numbers are limited at the systemic level. Additionally, HSC numbers in transplanted wild-type femurs did not increase beyond physiological levels when HSCs were mobilized from defective endogenous niches to the periphery, indicating that HSC numbers are also constrained at the local level. Our study demonstrates that HSC numbers are not solely determined by niche availability, thereby rewriting the long-standing model for the regulation of HSC numbers.