Influenza A virus uses the low pH in late endocytic vacuoles as a cue for penetration by membrane fusion. Here, we analyzed the pre-fusion reactions that prepare the core for uncoating after it has been delivered to the cytosol. We found that this priming process occurs in two steps that are mediated by the envelope-embedded M2 ion channel. The first weakens the interactions between the matrix protein, M1, and the viral ribonucleoprotein bundle. It involves a conformational change in a linker sequence and the C-terminal domain of M1 after exposure to pH below 6.5. The second step is triggered by pH < 6.0 and by the influx of K⁺ ions. It causes additional changes in M1 as well as a loss of stability in the vRNP bundle. Our results indicated that the switch from Na⁺ to K⁺ in maturing endosomes together with the decreasing pH are both needed to prime IAV cores for efficient uncoating and infection of the host cell.