Tolerogenic dendritic cells (Tol-DCs) are critical players in physiological tolerance. Moreover, they also play a role in immune regulation both in a pathophysiological context and when used as therapeutic tools in cell therapy strategies. Here, we describe a protocol to differentiate murine Tol-DCs from bone marrow precursors in vitro. Importantly, Tol-DCs actively suppress T cells stimulated with immunogenic allogeneic DCs. Indeed, Tol-DCs generated using this approach can be useful in studying and characterising the immunoregulatory pathways of Tol-DC and in developing Tol-DC-based cell therapy protocols using in vivo models.