The family of core binding factors includes the DNA-binding subunits Runx1-3 and the common non-DNA binding partner CBFβ. Runx1 and CBFβ are essential for the emergence of hematopoietic stem cells during fetal development, but not for stem cell maintenance during later ontogeny. Runx1 is also required for megakaryocyte differentiation, B cell development, and for the DN2 to DN3 transition in thymocyte development. Runx2/CBFβ are critical for normal osteogenesis, and Runx3 for CD4 silencing in CD8+ T cells, but their contribution to other steps of hematopoietic development is unknown. To examine the collective role of core binding factors in hematopoiesis, we generated a hypomorphic Cbfb allele (Cbfbrss). CBFβ protein levels were reduced by approximately 2–3 fold in fetuses homozygous for the Cbfbrss allele (Cbfbrss/rss), and 3–4 fold in fetuses carrying one hypomorphic and one knockout allele (Cbfbrss/−). Cbfbrss/rss and Cbfbrss/− fetuses had normal erythroid and B cell development, and relatively mild abnormalities in megakaryocyte and granulocyte differentiation. In contrast, T cell development was very sensitive to an incremental reduction of CBFβ levels: mature thymocytes were decreased in Cbfbrss/rss fetuses, and virtually absent in Cbfbrss/−fetuses. We next assessed the development of Cbfbrss/rss and Cbfbrss/− fetal liver progenitors after transplantation to irradiated adult recipients, in competition with wild-type (wt) bone marrow cells. Wt, Cbfbrss/rss and Cbfbrss/− fetal progenitors replenished the erythroid, myeloid and B cell compartments equally well. The overall development of Cbfbrss/rss T cells was preserved, although CD4 expression was derepressed in double negative thymocytes. In Cbfbrss/− chimeras, mature thymocytes were entirely derived from competitor cells. Furthermore, the developmental block in Cbfbrss/− progenitors was present at the earliest stages of T cell development within the DN1 (ETP) and DN2 subsets. Our data define a critical CBFβ threshold for normal T cell development, and they situate an essential role of core binding factors during the earliest stages of T cell development. In addition, early thymopoiesis appeared more severely affected by reduced CBFβ dosage than by the lack of Runx1 (Ichikawa et al., Nat Med 2004; Growney et al., Blood 2005), suggesting that Runx2/3 may contribute to core binding factor activity in the T cell lineage.