Aflatoxin (AF) B1 is a potent hepatotoxic, mutagenic, teratogenic mycotoxin and may cause immune suppression/dysregulation in humans and animals. Toxic effects of AFB1 on key mammalian immune cells (ie, leukocytes) needs to be mechanistically elucidated. In this study, along with the determination of AFB1’s LC50 for certain leukocytes, we analyzed the effect of naturally occurring levels of AFB1 on apoptosis/necrosis of neutrophils, lymphocytes, and monocytes from healthy young humans (20- to 25-year-old male), dogs (1- to 2-year-old Persian/herd breed), and cattle (1- to 2-year-old cattle). Leukocytes were incubated for approximately 24 hours with naturally occurring levels of AFB1 (10 ng/mL). Intracellular adenosine triphosphate (ATP) depletion and caspase-3/7 activity were then determined by luciferase-dependent bioluminescence (BL). Furthermore, the necrotic leukocytes were measured using propidium iodide (PI)-related flow cytometry. A significant decrease (24%-45%, 33.2% ± 2.7%) in intracellular ATP content was observed in AFB1-treated neutrophils, lymphocytes, and monocytes in all studied mammals. Also, with such a low level (10 ng/mL) of AFB1, BL-based caspase-3/7 activity (BL intensity) in all 3 tested mammalian leukocyte lineages was noticeably increased (∼>2-fold). Flow cytometry-based PI staining (for viability assay) of the AFB1-treated leukocytes showed slightly/insignificantly more increase of necrotic (PI+) neutrophils, lymphocytes, and monocytes in human, dogs, and cattle. Even though in vitro LC50s for AFB1’ (∼20,000-40,000 ng/mL) were approximately 2,000 to 4,000 times higher than background, these studies demonstrate leukocytes from human and farm/companion animals are sensitive to naturally occurring levels of AFB1. The observed in vitro ATP depletion and caspase activation in AFB1-exposed leukocytes can partially explain the underlying mechanisms of AFB1-induced immune disorders in mammals.