Haematopoietic stem and progenitor cells (HSPCs) derive from a subset of endothelial cells (ECs), known as haemogenic ECs by the process of endothelial-to-haematopoietic transition (EHT). Although many factors involved in EHT have been elucidated, we still have a poor understanding of the temporal regulation of this process. Mitochondrial-derived reactive oxygen species (ROS) have been shown to stabilise the hypoxia-inducible factor 1/2α (Hif1/2α) proteins, allowing them to positively regulate EHT. Here, we show a developmental delay in EHT and HSPC induction in a connexin (cx)41.8 (orthologous to mammalian CX40) gap junction mutant, in zebrafish. In mammalian cells, CX40 has been shown to localise to the mitochondria. We demonstrate that Cx41.8 is important for the correct temporal generation of mitochondrial ROS, which stabilise the Hif pathway, allowing for the subsequent specification of the haemogenic endothelium. Taken together, our data indicate that Cx41.8 governs the correct temporal induction of HSPCs.