Hematopoietic stem cells (HSCs) reside in specific microenvironments that facilitate their regulation through both internal mechanisms and external cues. Bone marrow endothelial cells (BMECs), which are found in one of these microenvironments, play a vital role in controlling the self-renewal and differentiation of HSCs during hematological stress. We previously showed that 27-hydroxycholesterol (27HC) administration of exogenous 27HC negatively affected the population of HSCs and progenitor cells by increasing the reactive oxygen species levels in the bone marrow. However, the effect of 27HC on BMECs is unclear. To determine the function of 27HC in BMECs, we employed magnetic-activated cell sorting to isolate CD31 BMECs and CD31 cells. We demonstrated the effect of 27HC on CD31 BMECs and HSCs. Treatment with exogenous 27HC led to a decrease in the number of BMECs and reduced the expression of adhesion molecules that are crucial for maintaining HSCs. Our results demonstrate that BMECs are sensitively affected by 27HC and are crucial for HSC survival.