In the bone marrow microenvironment, endothelial cells (ECs) play a pivotal role in regulating the production of both growth and inhibiting factors. They are held together by adherence molecules that interact with hematopoietic progenitor cells. The study of ECs in the hematopoietic stem cell niche is limited due to the lack of efficient protocols for isolation. In this protocol, we developed a two-step approach to extract bone marrow endothelial cells (BMECs) to unlock the challenges researchers face in understanding the function of the endothelial vascular niche in in-vitro studies.