Previously, we found a significant increase in TRAIL (TNFα-related apoptosis-inducing ligand) resistance of acute myeloid leukemia (AML) cells in multicellular aggregates in vitro that simulate the location of leukemia cells in the bone marrow. In this study, we tried to assess whether the increased resistance of the AML cells to the cytotoxic effect of TRAIL in multicellular aggregates in vitro can be associated with the launch of mechanisms mediated by cell differentiation or the acquisition of a pro-inflammatory phenotype. The phenotype of THP-1 cells in aggregated and disaggregated cultures was studied in comparison with the phenotype of a disaggregated THP-1 cell culture exposed to factors that induce monocyte-like differentiation or pro-inflammatory phenotype. It was found that an increase in the TRAIL resistance of the THP-1 cells in the aggregated culture was accompanied by the appearance of signs characteristic of monocyte-like differentiation and a pro-inflammatory phenotype. The results are of interest for developing approaches for suppressing TRAIL resistance of AML cells in human bone marrow.