In this study, we use competitive repopulation to compare the quality and frequency of stem cells isolated from mobilized blood with stem cells isolated from bone marrow (BM) in a mouse model. Lin− Sca-1+ c-Kit+(LSK) cells were harvested from control BM and peripheral blood of mice following granulocyte colony-stimulating factor (G-CSF) administration. LSK cells were used because of their resemblance to human CD34+ cells. We confirmed that transplantation of phenotypically defined mobilized peripheral blood (MPB) stem cells results in rapid recovery of blood counts. However, in vitro results indicated that LSK cells purified from MPB had lower cobblestone area-forming cell day 35 activity compared to BM. Additionally, evaluation of chimerism after co-transplantation of LSK cells purified from blood and BM revealed that MPB stem cells contained 25-fold less repopulation potential compared to BM stem cells. Competitive repopulating unit frequency analysis showed that freshly isolated MPB LSK cells have 8.8-fold fewer cells with long-term repopulating ability compared to BM LSK cells. Secondary transplantation showed no further decline in contribution of hematopoiesis relative to BM. We conclude that the reduced frequency of stem cells within the LSK population of MPB, rather than poorer quality, causes the reduced repopulation potential.