Human pluripotent stem cell (hPSC)-derived macrophages offer new opportunities to understand the role of development or tissue context in innate immune cell function. Immune responsiveness to pathogenic challenge is known to be impacted by a macrophage's history of prior exposure, as well as ontogeny and tissue context. Therefore, we explore the factors of in vitro derivation likely to influence macrophage phenotype and function, and highlight the gaps in, not only our understanding of macrophage development and function, but also in hPSC models that aim to mimic their in vivo counterparts. We developed a transcriptomic atlas to assess impact of ontogeny, experimental treatment and tissue residency on molecular phenotype. We revealed gaps in hPSC-macrophages, revealing several differences between hPSC-macrophages, monocyte-derived macrophages and primary cells. These differences include poor maturation in hPSC-macrophages in the absence of priming signals such as IFN, or repeated exposure to LPS. We demonstrate the requirement for priming in hPSC-macrophages and discover the importance of re-stimulation events in shaping macrophage activation. We further assess phenotypic heterogeneity in both monocyte- and hPSC-macrophages using single-cell sequencing. We demonstrate synchronised population responses to LPS activation and further provide evidence for priming in shaping macrophage responsiveness. These findings highlight that macrophages are shaped by prior activating, or priming, signals which can be recapitulated in the laboratory. Outcomes of this work are expected to improve routine macrophage derivation from hPSC sources, as exploitation of priming provides future opportunities to shape the quality of acute or long-term macrophage responsiveness for diverse applications.