We have characterized the fetal totipotent hematopoietic stem cell using a novel strategy that integrates physical analysis of cell properties and genetic analysis of in vivo developmental behavior. This approach allows the simultaneous isolation and in vivo characterization of any stem cell population. Using this procedure we demonstrate that a cell surface marker, recognized by monoclonal antibody AA4.1, defines 0.5%–1.0% of fetal liver tissue that contains the entire hierarchy of primitive hematopoietic cells. The AA4.1⁺ subpopulation includes multipotential in vitro progenitors, CFU-S cells, and lymphoid-myeloid stem cells that function to yield permanent and oligoclonal blood systems. Further fractionation of these cells by analysis of density, fibronectin binding, and surface antigen distribution has defined 0.1%–0.2% of fetal liver that contains the totipotent stem cell.