In the thymus, Notch signaling is essential for T lymphopoiesis, with Delta-like (Dll)4 uniquely involved in this process. However, using cocultures, either Dll4 or Dll1 were shown to support T lymphopoiesis. To address which Dll is more effective at inducing hematopoietic progenitor cells to give rise to T lineage cells in vitro, we generated OP9 cells expressing a series of incrementally discrete and equivalent levels of Dll1 or Dll4. In keeping with previous findings, OP9 cells expressing high levels of either Dll1 or Dll4 gave rise to T lineage cells with similar efficacy, and prevented the differentiation of B and myeloid-lineage cells. However, at limiting levels, Dll4 maintained its ability to inhibit B lineage choice and induce T lineage commitment and differentiation at lower levels than Dll1. This manifest property of Dll4 is evident despite lower levels of steady-state surface expression than Dll1 on OP9 cells. The heightened effectiveness of Dll4 over Dll1 also corresponded to the induction of Notch target genes, and inhibition of B and myeloid-specific transcription factors. Furthermore, we show that OP9 cells expressing levels of Dll4 equivalent to those present in thymic epithelial cells, as expected, gave rise to T lineage cells, but were also permissive for the differentiation of myeloid cells; whereas, still inhibiting B lymphopoiesis. Our findings show that Dll4 expressed at physiological levels on OP9 cells is functionally distinct from similarly expressed levels of Dll1, illustrating the unique properties of Dll4 in supporting the combined T lineage and specific myeloid-lineage outcomes that underpin its function within the thymus.