(A) EdU staining of runx1+23:mCherry embryos injected with either the calr3a, calr3b, or irf8 morpholinos identifies significant reduction in proliferating HSPCs at 3 dpf. Mean +/− s.d., One-way ANOVA with Dunnett’s multiple comparisons test; **P<0.01, ****P<0.0001. (B)(C) Single-cell mRNA-seq analysis of runx1+23+ FACS-purified cells from irf8 or control morphants reveals a population of stressed HSPCs that persist in the absence of macrophages and a population of cycling cells enriched in the control sample. (D) Embryonic HSPCs marked by surface Calreticulin exhibit higher levels of ROS. (E) ROS inhibition with diphenylene iodonium significantly reduces macrophage-HSPC interactions. Mean +/− s.d., Unpaired t test; *P<0.05. (F) Expression of il1b by heat shock rescues the effect of macrophage depletion on HSPC proliferation. Mean +/− s.d., One-way ANOVA with Sidak’s multiple comparisons test; *P<0.05, ****P<0.001.