The human monocytic THP-1 cell line is the most routinely employed in vitro model for studying monocyte-to-macrophage differentiation. Despite the wide use of this model, differentiation protocols using phorbol 12-myristate-13-acetate (PMA) or 1,25-dihydroxyvitamin D (1,25D) vary drastically between studies. Given that differences in differentiation protocols have the potential to impact the characteristics of the macrophages produced, we aimed to assess the efficacy of three different THP-1 differentiation protocols by assessing changes in morphology and gene- and cell surface macrophage marker expression. THP-1 cells were differentiated with either 5 nM PMA, 10 nM 1,25D, or a combination thereof, followed by a rest period. The results indicated that all three protocols significantly increased the expression of the macrophage markers, CD11b (p < 0.001) and CD14 (p < 0.010). Despite this, THP-1 cells exposed to 1,25D alone did not adopt the morphological and expression characteristics associated with macrophages. PMA was required to produce these characteristics, which were found to be more pronounced in the presence of 1,25D. Both PMA- and PMA with 1,25Ddifferentiated THP-1 cells were capable of M1 and M2 macrophage polarization, though the gene expression of polarization-associated markers was most pronounced in PMA with 1,25Ddifferentiated THP-1 cells. Moreover, the combination of PMA with 1,25D appeared to support the process of commitment to a particular polarization state.