The extracellular matrix plays a critical role in macrophage maturation. In this study, the HL60 cell line was used as a model of leukemic myeloid cell differentiation. We assessed the ability of HL60 cells cultured on fibronectin substratum prior to phorbol 12-myristate 13-acetate (PMA) induction to differentiate into terminally differentiated macrophages. Beside their distinctive macrophage morphology, they expressed antigen receptors CD14, TLR2, TLR4 and CD68, and displayed enhanced phagocytic activity and production of reactive oxygen species. Expression of CD13, CD33, CD15 and alpha-naphthyl-acetate esterase was also maintained, however, differentiated HL60 cells were HLA-DR and CD1a negative. Here, we describe the enhanced capacity of fibronectin-adherent HL60 cells to differentiate into macrophages in response to PMA.