Muscle mass differs by more than two-fold between healthy adult individuals of the same sex, age and height. Genetic factors account for 40 – 80% of these differences (1), however, specific genes responsible for the effects remain largely unknown. It also remains unclear to what extent these differences are due to the variation in the fibre number, determined prenatally, or fibre size, which can change over the course of a lifespan. The loss of muscle due to aging, known as sarcopenia, increases the risk of disability in the elderly 1.5 to 4.6 times (2). Understanding the mechanisms determining muscle mass and fibre numbers would help early assessment of the risk of sarcopenia when preventive interventions are more effective. The aim of this study was to shed light on genetic mechanisms determining muscle mass differences in a population of CFW outbred mice. The study was motivated by the findings of a genome-wide association analysis that a locus on chromosome X, encompassing Pou3f4, Cycl1 and Rps6ka6 genes, affected the weight of the glycolytic muscle (tibialis anterior, extensor digitorum longus (EDL), gastrocnemius and plantaris) in CFW mice (3). To follow that up, we carried out histological analyses in a subset of CFW EDL samples to explore the cellular mechanisms of the effect of that locus on muscle weight in male mice. We found that carriers of the weight-increasing allele had 10% more fibres (P<0.03) in EDL muscle, 1,091±92 (n=10), than the carriers of the decreasing allele, 988±95 (n=10), whereas we did not find differences in the cross-sectional area of muscle fibres. We then tested the hypothesis that Pou3f4 gene might be responsible for the effect on muscle fibre number. We carried out histochemical staining to compare the fibre numbers between Pou3f4-/- mice (n=7) and their wildtype littermates (n=5-7). The knockout mice had 21% reduction in EDL fibres, 847±85 vs 1,068±114 (P<0.01), and 22% reduction in fibres of oxidative soleus muscle, 591±144 vs 759±117 (P<0.04), compared to their wildtype littermates. We conclude that chromosome X locus affects muscle mass by influencing the number of muscle fibres. The Pou3f4 gene appears an underlying cause of the effect. Hence, the study identified a novel myogenic modifier that might be contributing to variability in muscle mass by determining fibre number.