Hematopoietic stem cells (HSC), the well-characterized adult stem cells both in the markers and function, show tremendous therapeutic potential. However, the level of hematopoietic stem cells in bone marrow is very low, which makes it difficult to work with. Based on cell surface markers and dye staining, HSC can be isolated from bone marrow and peripheral blood by using magnetic-activated cell separation and fluorescence-activated cell sorting. Over 40 years of research, many surface markers have been identified to purify mouse HSC, and CD34(-)LSK cells are regarded as the mostly used enrichment of HSC in mouse bone marrow. The purpose of this review is to provide insight into the advance of these markers, and isolation and purification of HSC.