Abstract Although Notch ligands are considered to activate signaling through direct cell-cell contact, the existence of soluble forms has been demonstrated. However, their roles remain controversial: soluble forms have been reported to mimic the biological activity of membrane-bound form, whereas other studies rather suggested an antagonistic activity toward their full-length counterparts. We previously observed that membrane-bound Delta4-expressing S17 stroma (mbD4/S17) reduced human CD34+CD38low cell proliferation and favored self-renewal. Here, we assessed the effects of a soluble form of Delta4 (solD4) by exposing CD34+CD38low cells to S17 feeders engineered to express solD4 (solD4/S17). In contrast to mbD4/S17, (a) solD4/S17 increased 10-fold cell production after 2 weeks, through enhanced cell proliferation, and (b) it did not preserve colony-forming cell and long-term culture-initiating cell potential of output CD34+ cells. mbD4 and solD4 appeared to also differ in their signaling. Indeed, mbD4, but not solD4, strongly activated both CSL (the nuclear mediator of Notch signaling) in Hela cells overexpressing Notch1 and transcription of some classic Notch target genes in CD34+CD38low cells. Furthermore, both biological effects and CSL activation elicited by mbD4 were strictly dependent upon the γ-secretase complex, whereas solD4 enhanced cell expansion in a partially γ-secretase-independent manner. Altogether, these results suggest that part of solD4 activity did not rely upon canonical Notch pathway. Disclosure of potential conflicts of interest is found at the end of this article.