Antibodies used in this study.

<div> Neutrophils are critical for host defense against fungi. However, the short life span and lack of genetic tractability of primary human neutrophils has limited <i>in vitro</i> analysis of neutrophil-fungal interactions. Human induced pluripotent stem cell (iPSC)-derived neutrophils (iNeutrophils) provide a genetically tractable system to study host defense responses of human neutrophils. Here, we show that deletion of the transcription factor GATA1 from human iPSCs results in iNeutrophils with improved antifungal activity against <i>Aspergillus fumigatus</i>. GATA1-knockout (KO) iNeutrophils have increased maturation, antifungal pattern recognition receptor expression and have improved neutrophil effector functions compared to wild-type iNeutrophils. iNeutrophils also show a shift in their metabolism following stimulation with fungal β-glucan to the pentose phosphate pathway (PPP), similar to primary human neutrophils. Furthermore, we show that deletion of the integrin CD18 attenuates the ability of GATA1-KO iNeutrophils to kill <i>A. fumigatus</i> but is not necessary for the metabolic shift<i>.</i> Collectively, these findings support iNeutrophils as a robust system to study human neutrophil antifungal immunity and has identified specific roles for CD18 in the defense response<i>.</i> </div>