Hematopoietic stem and progenitor cell (HSPC) differentiation relies on cytokine receptors such as Flk2 and IL7R to receive instructive signals from the environment (Zhang and Lodish, 2008). For example, we have previously shown that Flk2 is intrinsically required on HSPCs for both myeloid and lymphoid reconstitution, as demonstrated by the reduced capacity of Flk2−/− HSCs to generate myeloid and lymphoid progeny, including traditional circulating lymphoid cells, upon transplantation into a WT host (Beaudin et al., 2014). More recently, we demonstrated an extrinsic requirement of IL7Rα in the generation of eosinophils from WT HSCs, as demonstrated by a reduced capacity of WT HSCs to generate eosinophils upon transplantation into an IL7Rα−/− host (Cool et al., 2020). Therefore, we were curious whether the deficiency in TLCs we observed in Flk2−/−, IL7Rα−/− and FIDKO mice (Fig. 2) is due to the cell-intrinsic lack of receptors or due to changes to surrounding cells. To begin to answer this question, we transplanted WT, Flk2−/−, IL7Rα−/− or FIDKO HSCs into sublethally irradiated WT hosts (Fig. 3A). Under such transplantation conditions, host cells are partially, but not completely, depleted (Boyer et al., 2019), leading to robust and long-term HSC engraftment (Fig. S3). We first examined donor chimerism of circulating lymphoid cells. As expected, peripheral blood B cell donor chimerism was significantly lower from Flk2−/− donor HSCs, IL7Rα−/− HSCs and FIDKO HSCs compared with WT HSCs (Fig. 3B); similar results were observed for peripheral blood T cells (Fig. S2B), whereas granulocyte/macrophage (GM) reconstitution was significantly impaired only from Flk2−/− donor HSCs (Fig. S3A). When examining TLCs, we observed only donor chimerism of IL7Rα−/− and FIDKO HSCs was significantly impaired, whereas Flk2 deficiency alone did not result in significant differences (Fig. 3C-F′; Fig. S1C,C″). A limitation of donor chimerism is that this measure relies on host cell numbers and therefore makes it more difficult to determine the intrinsic requirement of these receptors because the number of host cells changes dynamically over time post-conditioning (Boyer et al., 2019). To overcome this limitation, we quantified the absolute cellularity of donor-derived cells with a bead-based method that allows for simultaneous flow cytometric analysis and counting, which is purely a measure of donor cells (Boyer et al., 2019; Rajendiran et al., 2020; Poscablo et al., 2021). Quantification of absolute cellularity of donor-derived cells between WT and knockout HSCs revealed that reconstitution of B1a cells (Fig. 3C′) and MZBs (Fig. 3D′) was also significantly impaired by the loss of Flk2 alone. Regardless of quantification method, deletion of either IL7Rα alone, or both IL7Rα and Flk2, led to impaired reconstitution of all TLCs examined (Fig. 3C′-F′). This was intriguing as we did not observe significant reduction of Tregs in IL7Rα−/− at steady state (Fig. 2F). This is likely due to the stress of transplantation, which reveals more dynamic requirement of IL7Rα – we previously observed a similar trend when we compared GM at steady state in Flk2−/− mice to transplantation of Flk2−/− HSCs (Beaudin et al., 2014). These data suggest that IL7Rα is required cell intrinsically for TLC reconstitution, that Flk2 cannot compensate for this requirement and that IL7Rα is not capable of fully compensating for the loss of Flk2.