Myogenic differentiation, muscle stem cell functionality, and regeneration of skeletal muscle are cellular processes under tight control of various signaling pathways. Here, we investigated the role of non-canonical NF-κB signaling in myogenic differentiation, muscle stem cell functionality, and regeneration of skeletal muscle. We stimulated non-canonical NF-κB signaling with an agonistically acting antibody of the lymphotoxin beta receptor (LTβR). Interestingly, we found that stimulation of non-canonical NF-κB signaling through the LTβR agonist impairs myogenic differentiation, muscle stem cell function, and regeneration of skeletal muscle. Furthermore, we show that stimulation of non-canonical NF-κB signaling by the LTβR agonist coincides with activation of canonical NF-κB signaling. We suggest a direct crosstalk between canonical and non-canonical NF-κB signaling during myogenic differentiation which is required for proper myogenic differentiation and thereby regeneration of skeletal muscle.