The mechanisms that regulate skeletal muscle development involve the coordinated activity of transcription factors (TFs) and the precise timing of gene expression patterns. NF-Y is a heterotrimeric TF with a pioneer role in the transcriptional and epigenetic regulation of promoters containing the CCAAT-box. NF-Y activates the expression of various genes related to the cell cycle, particularly genes of the G2/M phase. NF-YA, the regulatory DNA-binding subunit of the complex, is expressed in proliferating myoblasts and down-regulated during terminal differentiation. The NF-YA gene encodes for two alternatively spliced isoforms, namely NF-YAs and NF-YAl, which are not functionally identical. Using mouse C2C12 cells, we provide evidence of a different role for NF-YA variants in the myogenic program. While NF-YAs enhances myoblasts proliferation, NF-YAl boosts their differentiation by up-regulating the transcription of novel target genes, among which Mef2D, Sixs and Cdkn1C, which are known to be involved in the differentiation program. We further demonstrate that NF-YA is expressed in resident stem cells (SCs) and the two isoforms are transcribed at different levels during SCs activation and differentiation. The inhibition of NF-Y activity impairs both proliferation and differentiation of SCs and the overexpression of the two NF-YA isoforms differentially affects their fate.