AbstractMonocytic differentiation is coordinated through the ordered activation of multiple signalling pathways, controlling transcription of specific subsets of genes that regulate the development of the mature phenotype. To identify key transcription factors involved in this process, we used the human monoblastic U‐937 cell line as a model of monocytic differentiation. U‐937 cells can be differentiated by treatment with all‐trans retinoic acid (ATRA) and 1,25α‐dihydroxycholecalciferol (VitD3), resulting in G0/G1‐arrested cells expressing monocytic surface markers. We have previously shown that ATRA‐induced differentiation and cell cycle arrest specifically requires Stat1 activation, through phosphorylation of tyrosine 701 and serine 727. In this report, we used U‐937 cells expressing phosphorylation‐deficient mutants of Stat1 (Stat1Y701F and Stat1S727A) to determine myeloid‐specific transcription factors that are activated downstream of Stat1 during induced monocytic differentiation. We demonstrate that ATRA‐induced upregulation of Stat2, ICSBP/IRF8 and C/EBPɛ, key transcription factors linked to myelomonocytic differentiation, is selectively impaired in cells expressing mutant Stat1. In contrast, ATRA‐induced expression of PU.1, C/EBPα, C/EBPβ and IRF‐1 was unaffected. Taken together, our data suggest that ATRA‐induced regulation of Stat2, ICSBP and C/EBPɛ is dependent on active Stat1, and that a failure to correctly regulate these transcription factors is associated with the inhibition of monocytic differentiation.