Satellite cells (SCs) are essential for postnatal muscle growth and regeneration, however, their expansion potential in vitro is limited. Recently, hypoxia has been used to enhance proliferative abilities in vitro of various primary cultures. Here, by isolating SCs from single mouse hindlimb skeletal myofibers, we were able to distinguish two subpopulations of clonally cultured SCs (Low Proliferative Clones--LPC--and High Proliferative Clones--HPC), which, as shown in rat skeletal muscle, were present at a fixed proportion. In addition, culturing LPC and HPC at a low level of oxygen we observed a two fold increased proliferation both for LPC and HPC. LPC showed higher myogenic regulatory factor (MRF) expression than HPC, particularly under the hypoxic condition. Notably, a different myogenic potential between LPC and HPC was retained in vivo: green fluorescent protein (GFP)+LPC transplantation in cardiotoxin-injured Tibialis Anterior led to a higher number of new GFP+muscle fibers per transplanted cell than GFP+HPC. Interestingly, the in vivo myogenic potential of a single cell from an LPC is similar if cultured both in normoxia and hypoxia. Therefore, starting from a single satellite cell, hypoxia allows a larger expansion of LPC than normal O(2) conditions, obtaining a consistent amount of cells for transplantation, but maintaining their myogenic regeneration potential.