Calcium signaling plays key roles during human myoblast differentiation. The calcium-dependent phosphatase Calcineurin is essential for myoblast differentiation and muscle regeneration. NFAT transcription factors (Nuclear Factor of Activated T-cell) are the major Calcineurin targets. By investigating their expression and their role, I found that three NFAT are present in human primary myoblasts, NFATc1, NFATc3 and NFATc4. Surprisingly, while their expression increased during differentiation, NFATc1 is more expressed in myotubes and NFATc4 in the reserve cells. Although NFATc3 is expressed in both cell types but does not seem to have a particular role during myoblast differentiation. When NFATc1 or NFATc4 expression is impaired by siRNAs, differentiation is affected. The expression of late differentiation markers, Myosin HC and STIM1L, is decreased and myotube formation is impaired, but differently. Indeed, NFATc1 knockdown strongly reduced the number and the surface of myotubes formed, NFATc4 knockdown increased myotube surface and reduced the reserve cells pool.