Protocol to generate human T cell progenitors that repopulate the thymus.

Human pluripotent stem cells (hPSCs) provide a powerful platform for generating T cell progenitors and investigating hematopoietic stem cell (HSC)-independent T cell development. Here, we present a protocol for the directed differentiation of hPSCs to generate early progenitors of human T cells that repopulate the thymus long term and gradually develop into mature T cells in vivo. The protocol includes primary hematopoietic differentiation of hPSCs in defined culture conditions, a secondary differentiation step to prime T lymphopoiesis, and mouse transplantation procedures. For complete details on the use and execution of this protocol, please refer to Philonenko et al.1.