Evidence for the lineage relationship between embryonic and adult hematopoietic stem cells (HSCs) in the mouse is primarily indirect. In order to study this relationship in a direct manner, we expressed the tamoxifen-inducible Cre-EFT recombinase under the control of the stem cell leukemia (Scl) stem-cell enhancer in transgenic mice (HSC-SCL-Cre-ERT). To determine functionality, HSC-SCL-Cre-ERT transgenics were bred with Cre reporter mice. Flow cytometric and transplantation studies revealed tamoxifen-dependent recombination occurring in more than 90% of adult long-term HSCs, whereas the targeted proportion within mature progenitor populations was significantly lower. Moreover, the transgene was able to irreversibly tag embryonic HSCs on days 10 and 11 of gestation. These cells contributed to bone marrow hematopoiesis 5 months later. In order to investigate whether the de novo HSC generation is completed during embryogenesis, HSC-SCL-Cre-ERT-marked fetal liver cells were transplanted into adult recipients. Strikingly, the proportion of marked cells within the transplanted and the in vivo-remaining HSC compartment was not different, implying that no further HSC generation occurred during late fetal and neonatal stages of development. These data demonstrate for the first time the direct lineage relationship between midgestation embryonic and adult HSCs in the mouse. Additionally, the HSC-SCL-Cre-ERT mice will provide a valuable tool to achieve temporally controlled genetic manipulation of HSCs. (c) 2005 by The American Society of Hematology.