Satellite cells are key cells for post-natal muscle growth and regeneration and they play a central role in the search for therapies to treat muscle injuries. In this study the proliferation and differentiation capacity of muscle progenitor cells was studied in 2D and 3D cultures with collagen type I and Matrigel, which contain the niche factors laminin and collagen type IV. Muscle progenitor cells were cultured to induce proliferation and differentiation in collagen- or Matrigel-coated surfaces (2D) or in gels (3D). In the 2D cultures, muscle progenitor cells proliferated faster in Matrigel than in collagen. The numbers of Pax7(+) and MyoD(+) cells were also significantly higher in Matrigel than in collagen. During differentiation, muscle progenitor cells formed more and larger MyoD(+) and myogenin(+) myotubes in Matrigel. In the 3D cultures, muscle progenitor cells in Matrigel expressed higher mRNA levels of MyoD and myogenin, and formed elongated myotubes expressing myogenin and myosin. In collagen gels, the myotubes were short and rounded. In conclusion, muscle progenitor cells, both in 2D and 3D, lose their differentiation capacity in collagen but not in Matrigel. Although Matrigel contains growth factors, our results indicate that the kind of biomaterial steers the maintenance of the myogenic potential and their proper differentiation to achieve optimal skeletal muscle restoration.