Filarial nematode parasites enter their definitive host via the bite of an insect vector at the developmentally arrested iL3 larvae stage. Exit from iL3, allowing growth to resume to reproductive adulthood, occurs in the favorable conditions provided by the mammalian host. Ligand-activated DAF-12 is considered a key factor of the development of filarial nematodes establishing parasite infection, but the natural ligands responsible for DAF-12 activation remain to be determined. We have recently shown that in vitro development of the dog Heartworm Dirofilaria immitis can be induced by different steroids of parasitic or mammalian origin, which activate also the nuclear receptor DAF‑12 (Long T et al. Sci Rep. 2020). With the use of a luciferase gene reporter assay, we observed that sera from different mammals, including dog which is the D. immitis host but also human, mouse, sheep, pig and cow, can activate DimDAF-12. Interestingly, Fetal Bovine Serum (FBS) but not charcoal stripped FBS, stimulated DimDAF-12 activity. As charcoal stripping removes most of the lipidic compounds from serum, this result suggests that DAF-12 activation occurs through lipidic ligands present in bovine serum. Intriguingly, mammalian sera also activated DAF-12 from the filarial nematode Brugia malayi (Bma), but did not activate the hookworm Haemonchus contortus (Hco) or Caenorhabditis elegans (Cel) DAF-12, suggesting that DAF-12 of filarial nematodes are specifically hypersensitive to mammalian sera. Remarkably, we observed that Dim and BmaDAF-12 exhibit much higher affinity for cholestenoic acid, a bile acid precursor present in mammalian serum, than Hco and CelDAF-12. Altogether, our data suggest that filarial nematode parasites have evolved to sense very low concentration of host steroids in order to resume their development at proper place and time.