Early responses to invading pathogens and to non-microbial danger signals are mediated by different innate immune and parenchymal tissue cells, which are able to respond to a variety of pathogen- and danger-associated molecular patterns. In most if not all instances, innate immune responses to a given molecule are not uniquely confined to one responding cell type, but instead involve the engagement of different cells with intrinsically distinct properties. In this Review, we discuss the molecular basis of the differentiation of myeloid cells, which is controlled by transcription factors, transcriptional co-regulators and post-transcriptional mechanisms, and examine how the functional specification of the resulting mature immune cells of the myeloid lineage affects their response to danger signals.