The discovery that highly pathogenic avian influenza A(H5N1) virus exhibits a strong tropism for the bovine mammary gland1–4 represents a major shift in our understanding of influenza A virus host range and tissue specificity. We conducted a comprehensive series of experimental studies with influenza A(H5N1) B3.13 genotype in lactating dairy cattle to address several key questions related to the viral dose required to establish infection, routes of exposure that lead to transmission, and factors contributing to the morbidity and mortality observed on farms. We demonstrate that intramammary exposure to as few as 10 TCID50 is sufficient to establish robust infection, shedding of high viral titers in milk, and clinical mastitis. Despite evidence of such a low infectious dose, we were unable to recapitulate transmission to sentinel cows via contaminated milking equipment and close contact with infected animals under experimental conditions. High-dose intramammary exposure to influenza A(H5N1) drives severe clinical outcomes and mortality observed in dairy cows on-farm, while respiratory and oral exposure are less likely to establish productive infection and associated morbidity. This study challenges current hypotheses of influenza A(H5N1) transmission on dairy farms5,6, raising important questions about potential agent, host, or environmental cofactors that contribute to the spread of the virus.