We created parabiotic mice, joining ROSA26 and PeP3^b animals, to study the trafficking of hematopoietic stem cells (HSCs) from marrow to blood and their return to marrow. The transfer of HSCs was assayed by secondary marrow transplantation and was 1.0% to 2.5% after 3, 6, 8, and 12 weeks. Thus, HSC homeostasis is primarily maintained by the retention of stem cells derived from replication events within the marrow, not the homing and engraftment of HSCs from the circulation. Of interest, the phenotypes of marrow progenitors and granulocytes were similar to those for HSCs, implying that the marrow functions as an intact compartment where differentiating cells derive from endogenous HSC. In contrast, 50% of splenic granulocytes and progenitor cells derived from the parabiotic partner, suggesting splenic progenitor cells were in constant equilibrium with progenitors in blood. In additional studies, animals were exposed to granulocyte–colonystimulating factor (G-CSF) and stem cell factor at days 17 to 20 of parabiosis and were studied 3 weeks later; 10.1% of marrow HSCs derived from the parabiotic partner. These data imply that HSCs, mobilized to the blood in response to cytokine exposure, are destined to later return to marrow, an observation that supports the concept that the mobilized peripheral blood stem cells used in clinical transplantation function physiologically.