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Nodal and its co-receptors of the EGF-CFC family are essential during chordate development, both to maintain pluripotent progenitor cells and to subsequently coordinate their allocation to the different germ layers. How these distinct Nodal activities are kept in balance is unknown. Previous work in our laboratory established that the Nodal proprotein must be cleaved by proprotein convertases (PC) to induce itself on time in the mouse epiblast. However, expression of a knock-in allele encoding a PC-resistant mutant Nodal precursor (Nr) is sufficient to induce a subset of Nodal target genes during gastrulation, including Fgf4 in the epiblast, and Bmp4 in extraembryonic ectoderm (ExE). Here, I extend these findings by showing that these NodalNr/Nr embryos also partially maintain a panel of trophoblast stem cell (TSC) markers. To better define how Nodal maintains pluripotency and how it might signal to TSCs in the ExE, and as a means to genetically uncouple Nodal production from processing, I treated cultured TSCs and NodallacZ/lacZ embryonic stem cells (ESCs) with recombinant Nodal or with cleavage-resistant mutant Nodal precursor. I show that recombinant mature Nodal stimulates Smad2 phosphorylation in both ESCs and TSCs, even though the latter do not express Nodal coreceptors of the EGF-CFC family. In contrast, recombinant Nr failed to activate the canonical Smad pathway in either cell type, and hence preferentially stimulated Jnk and p38 MAPK signaling. Yet, none of these activities were sufficient to promote self-renewal of mouse ESCs or TSCs under any of the culture conditions examined. Using lentiviral overexpression of constitutively active receptors with mutations in the Smad2,3 binding site, I observed that TSC stemness apparently can be maintained independently of Smad2,3 activation. These results suggest that mature Nodal or its unprocessed precursor likely promote the self-renewal of TSCs in vivo through a non-canonical, Smad-independent signaling pathway, but that available recombinant proteins for unknown reasons cannot fully mimic this activity in vitro, at least under the conditions examined.