Recent findings have shown that amniotic fluid (AF) could be a putative new source of multipotent stem cells (SC). We investigated whether these human SC could efficiently differentiate into myogenic lineage in vitro and integrate in vivo skeletal muscle in severe combined immunodeficiency (SCID) mice. C/kit immunomagnetic-sorted AF (AF c/kit+) SC were characterized by immunocytochemistry and Southern blotting for myogenic markers (desmin, MyoD). In vitro, AF c/kit+ SC phenotypic conversion into myogenic cells was assayed by myogenic-specific induction media. AF c/kit+ SC without ex vivo manipulation were transplanted into the tibialis anterior (TA) of (SCID) mice. Acquisition of a myogenic-like phenotype (desmin, MyoD) in AF c/kit+ SC was observed after culture in myogenic-specific induction media. In vivo, transplanted AF c/kit+ SC showed an engraftment in the skeletal muscle of SCID mice, but with unexpected tubular glandular tissue-like differentiation. Importantly, no immuno-rejection, inflammatory response or tumorigenicity of these cells was found. Within these experimental conditions, AF c/kit+ SC were able to differentiate into myogenic cells in vitro, but not in vivo after their transplantation into the skeletal muscle of SCID mice. Because AF c/kit+ SC survived and differentiated into tubular gland-like cells after their transplantation in the TA, an ex vivo engagement in myogenic pathway prior their transplantation could favor their differentiation into myogenic cells in vivo.