Influenza virus infections are a major public health concern and cause significant morbidity and mortality worldwide. Current vaccines are effective but strain specific due to their focus on the immunodominant globular head domain of the hemagglutinin (HA). It has been hypothesized that sequential exposure of humans to hemagglutinins with divergent globular head domains but conserved stalk domains could refocus the immune response to broadly neutralizing epitopes in the stalk. Humans have preexisting immunity against H1 (group 1 hemagglutinin), and vaccination with H5 HA (also group 1)-which has a divergent globular head domain but a similar stalk domain-represents one such sequential-exposure scenario. To test this hypothesis, we used novel reagents based on chimeric hemagglutinins to screen sera from an H5N1 clinical trial for induction of stalk-specific antibodies by quantitative enzyme-linked immunosorbent assay (ELISA) and neutralization assays. Importantly, we also investigated the biological activity of these antibodies in a passive transfer in a mouse challenge model. We found that the H5N1 vaccine induced high titers of stalk-reactive antibodies which were biologically active and protective in the passive-transfer experiment. The induced response showed exceptional breadth toward divergent group 1 hemagglutinins but did not extend to group 2 hemagglutinins. These data provide evidence for the hypothesis that sequential exposure to hemagglutinins with divergent globular head domains but conserved stalk domains can refocus the immune response toward the conserved stalk domain. Furthermore, the results support the concept of a chimeric hemagglutinin universal influenza virus vaccine strategy that is based on the same principle.