Background <p>Given the high relevance of human cardiac valve disease, recent research aims to differentiate human induced pluripotent stem cells (hiPSCs) into valve endothelial-like cells (VELCs) and, through endothelial-to-mesenchymal transition (EndMT), into valve interstitial-like cells (VILCs).</p> Methods <p>Here, we modified a 2D differentiation protocol demonstrating that VEGF can serve as the sole driver for differentiating cardiac progenitor cells (CPCs) into VELCs. Next, we utilized the so-called GiWi protocol (inhibition of glycogen synthase kinase, followed by inhibition of the Wnt pathway) to derive VELCs from 3D endocardial spheres. To this aim, hiPSCs were first differentiated into cardiac progenitor (CP) spheres using CHIR99021 (12 µM) and IWP2 (5 µM). Subsequent treatment with E8 medium containing high-dose FGF2 (100 ng/ml) resulted in endocardial spheres enriched for VELCs. For EndMT induction, endocardial spheres were MACS-sorted for PECAM1⁺ VELCs and transdifferentiated into ACTA2⁺/CDH5⁻ VILCs using TGFβ1 (200 ng/ml).</p> Results <p>Using VEGF as main driver to differentiate CPCs into VELCs in 2D, the generated VELCs appeared stable over time, can be maintained in vitro and transdifferentiated into ACTA2⁺ VILCs using FGF2 (100 ng/ml) and TGFβ1 (50 ng/ml). Besides, our novel 3D differentiation protocol yielded endocardial spheres, which were highly enriched with VELCs, as shown by the expression of GATA4 (≈ 83%), PECAM1 (≈ 69%), and nuclear NFATC1 (≈ 76%), along with typical functional characteristics such as network formation, LDL uptake and the ability to undergo EndMT.</p> Conclusions <p>Overall, we developed a 2D differentiation protocol that produces stable VELCs and a high percentage of VILCs upon EndMT induction. We also established a new, efficient, and cost-effective protocol for the 3D differentiation of endocardial spheres to better mimic a physiologically relevant environment, thereby enabling improved maturation.</p>