B1 lymphocytes provide a rapid antibody response in body cavities and mucosa. They are generated during foetal development and self-renew to maintain the adult population, but their developmental origin has been controversial. Although B1 progenitor cells reside in the foetal liver, the ability of transplanted foetal liver haematopoietic stem cells (HSCs) to repopulate B1 cells has provided contradictory results. Now, Momoko Yoshimoto, Andrea Ditadi and colleagues use Rbpj−/− mouse embryonic stem cells (mESCs), which have a Notch signalling deficiency crucial for HSC development, to understand whether B1 cell generation is Notch (and therefore HSC) dependent. Pre-B lymphocytes can be differentiated in vitro from Rbpj−/− mESCs, and the transplantation of pre-B lymphocytes into irradiated mice shows that Notch-deficient cells can mature into B1a and B1b, but not B2, cells. Next, the authors inhibit Notch signalling in ex vivo embryonic endothelial cells and reveal that B-cell lineages develop independently of Notch signalling. Conversely, over-activation of the Notch signalling pathway indicates that Notch signalling dose determines B2 or T-cell specification from precursors. Taken together, these data demonstrate that the development of B1 cells can occur in the absence of Notch signalling and potentially independently of HSC specification.