The hierarchical relationship of skeletal muscle-derived multipotent stem cells sorted as CD34(+)/CD45(-) (Sk-34) and CD34(-)/CD45(-) (Sk-DN) cells, which have synchronized reconstitution capacities for blood vessels, peripheral nerves, and muscle fibers, was examined. Expression of Sca-1 and CD34 (typical state of freshly isolated Sk-34 cells) in Sk-DN cells was examined using in vitro culture and in vivo cell implantation. Sk-DN cells sequentially expressed Sca-1 and CD34 during cell culture showing self-maintenance and/or self-renewal-like behavior, and are thus considered hierarchically upstream of Sk-34 cells in the same lineage. Sk-34 and Sk-DN cells were further divided into small and large cell fractions by cell sorting. Immunocytochemistry using anti-Pax7 was performed at the time of isolation (before culture) and revealed that only 1% of cells in the large Sk-DN cell fraction were positive for Pax7, while Sk-34 cells and 99% of Sk-DN cells were negative for Pax7. Therefore, putative satellite cells were possibly present in the large Sk-DN cell fraction. However, serial analysis of Pax7 expression by RT-PCR and immunocytochemistry for single and 2 to >40 clonally proliferated Sk-34 and Sk-DN cells revealed that both cell types expressed Pax7 after several asymmetric cellular divisions during clonal-cell culture. In addition, production of satellite cells was seen after muscle fiber formation following Sk-34 or Sk-DN cell transplantation into damaged muscle, and even in the nonmuscle tissue environment (beneath the renal capsule). Thus, Sk-DN cells are situated upstream of Sk-34 cells and both cells can produce Pax7+ cells (putative satellite cells) after cellular division.