Human influenza virus rapidly accumulates mutations in its major surface protein hemagglutinin (HA). The evolutionary success of influenza virus lineages depends on how these mutations affect HA's functionality and antigenicity. Here we experimentally measure the effects on viral growth in cell culture of all single amino-acid mutations to the HA from a recent human H3N2 influenza virus strain. We show that mutations that are measured to be more favorable for viral growth are enriched in evolutionarily successful H3N2 viral lineages relative to mutations that are measured to be less favorable for viral growth. Therefore, despite the well-known caveats about cell-culture measurements of viral fitness, such measurements can still be informative for understanding evolution in nature. We also compare our measurements for H3 HA to similar data previously generated for a distantly related H1 HA, and find substantial differences in which amino acids are preferred at many sites. For instance, the H3 HA has less disparity in mutational tolerance between the head and stalk domains than the H1 HA. Overall, our work suggests that experimental measurements of mutational effects can be leveraged to help understand the evolutionary fates of viral lineages in nature -but only when the measurements are made on a viral strain similar to the ones being studied in nature.influenza virus | hemagglutinin | deep mutational scanning | antigenic drift | epistasis S easonal H3N2 influenza virus evolves rapidly, fixing 3 to 4 amino-acid mutations per year in its hemagglutinin (HA) surface protein (1, 2). Many of these mutations contribute to the rapid antigenic drift that necessitates frequent updates to the annual influenza vaccine (3). This evolution is further characterized by competition and turnover among groups of strains called clades bearing different complements of mutations (4-8). Clades vary widely in their evolutionary success, with some dying out soon after emergence and others going on to take over the virus population. Several lines of evidence indicate that successful clades have higher fitness than clades that remain at low frequency (4-6, 9). A key goal in the study of H3N2 evolution is to identify the features that enable certain clades to succeed as others die out.Two main characteristics distinguish evolutionarily successful clades from their competitors: greater antigenic change, and efficient viral growth and transmission. In principle, experiments could be informative for identifying how mutations affect these features. Most work on influenza evolution to date has utilized experimental data to assess the antigenicity of circulating strains (11)(12)(13)(14)(15)(16). However, the non-antigenic effects of mutations also play an important role (5,7,9,17). Specifically, due to influenza virus's high mutation rate (18)(19)(20) and lack of intra-segment recombination (21), deleterious mutations become linked to beneficial ones. The resulting accumulation of deleterious mutations can affect non-antigenic properties central...