ABSTRACT Human leukemia cell line (HL-60) is an alternative to primary neutrophils in research studies. However, because HL-60 cells proliferate in an incompletely differentiated state, they must undergo differentiation before they acquire the functional properties of neutrophils. Here we provide evidence of swarming and chemotaxis in HL-60 neutrophil-like cells using precision microfluidic assays. We found that dimethyl sulfoxide (DMSO) differentiated HL-60 cells have larger size, increased length, and lower ability to squeeze through narrow channels compared to primary neutrophils. They migrate through tapered microfluidic channels slower than primary neutrophils, but faster than HL60s differentiated by other protocols, e.g. using all-trans retinoic acid. We found that differentiated HL-60 can swarm toward zymosan particle clusters, though they display disorganized migratory patterns and produce swarms of smaller size compared to primary neutrophils.