Basement membrane (BM) component deposition is closely linked to the establishment of cell polarity. Previously, we showed that Prickle1 is crucial for BM deposition and cell polarity events in tear duct elongation. To gain a deeper understanding of the intimate relationship between BM formation and cell polarity, we generated induced pluripotent stem cells (iPSCs)-derived embryoid bodies (EBs) with a basement membrane separating the visceral endoderm (VE) and inner EB cell mass. We found that Prickle1 was highly expressed in VE of the normal EBs, and the Prickle1 mutant EBs displayed severely impaired BM. Notably, the formation of the basement membrane appeared to rely on the proper microtubule network of the VE cells, which was disrupted in the Prickle1 mutant EBs. Moreover, disruption of vesicle trafficking in the VE hindered BM secretion. Furthermore, reintroducing Prickle1 in the mutant EBs completely rescued BM formation but not the apicobasal cell polarity of the VE. Our data, in conjunction with studies by others, highlight the conserved role of Prickle1 in directing the secretion of BM components of the VE cells during embryonic germ layer differentiation, even in the absence of established general polarity machinery. Our study introduces a novel system based on iPSCs-derived EBs for investigating cellular and molecular events associated with cell polarity.