Antigen-specific CD4(+) T cells are essential for effective virus-specific host responses, with recent human challenge studies (in volunteers) establishing their importance for influenza A virus (IAV)-specific immunity. However, while many IAV CD4(+) T cell epitopes have been identified, few are known to stimulate immunodominant CD4(+) T cell responses. Moreover, much remains unclear concerning the major antigen(s) responded to by the human CD4(+) T cells and the extents and magnitudes of these responses. We initiated a systematic screen of immunodominant CD4(+) T cell responses to IAV in healthy individuals. Using in vitro expanded-multispecificity IAV-specific T cell lines and individual IAV protein antigens produced by recombinant vaccinia viruses, we found that the internal matrix protein 1 (M1) and nucleoprotein (NP) were the immunodominant targets of CD4(+) T cell responses. Ten epitopes derived from M1 and NP were definitively characterized. Furthermore, epitope sequence conservation analysis established that immunodominance correlated with an increased frequency of mutations, reflecting the fact that these prominent epitopes are under greater selective pressure. Such evidence that particular CD4(+) T cells are important for protection/recovery is of value for the development of novel IAV vaccines and for our understanding of different profiles of susceptibility to these major pathogens. Importance: Influenza virus causes half a million deaths annually. CD4(+) T cell responses have been shown to be important for protection against influenza and for recovery. CD4(+) T cell responses are also critical for efficient CD8(+) T cell response and antibody response. As immunodominant T cells generally play a more important role, characterizing these immunodominant responses is critical for influenza vaccine development. We show here that the internal matrix protein 1 (M1) and nucleoprotein (NP), rather than the surface proteins reported previously, are the immunodominant targets of CD4(+) T cell responses. Interestingly, these immunodominant epitope regions accumulated many mutations over time, which likely indicates increased immune pressure. These findings have significant implications for the design of T cell-based influenza vaccines.