Individuals with Trisomy 21 (T21) exhibit numerous hematological abnormalities, including reductions in numbers of circulating B and T lymphocytes. To elucidate molecular mechanisms underlying these phenotypes, we differentiated human isogenic disomic and trisomic pluripotent cells, and observed that trisomic cells showed defects in B cell, but not T cell differentiation. Global gene expression of differentiated, trisomic B cells revealed reduced expression of genes encoding endothelin signaling components, namely the Endothelin Receptor B (EDNRB), and its ligand Endothelin1 (EDN1). Depletion of EDNRB mRNA in cord blood-derived CD34+ cells led to defective B cell differentiation, supporting a hypothesis that low EDNRB expression in T21 contributes to intrinsic lymphoid defects. Further evidence for the role of the EDNRB pathway in B cell differentiation was obtained through CRISPR/Cas9 gene targeting in disomic and trisomic iPS cells. Knockout of EDNRB in both cell backgrounds reduced the capacity for B cell differentiation. Collectively, this work identifies downregulation of EDNRB as a causative factor for impaired B lymphocyte generation in trisomic cells, which may contribute to defects in immune function associated with T21. Furthermore, a novel role for endothelin signaling in regulation of B cell development has been identified