Human pluripotent stem cells (hPSCs) were utilized to gain insight into the Wnt pathway, a developmentally important signaling pathway and to generate vocal fold epithelia, a hard to derive tissue for in vitro barrier characterization studies. Additionally, phenotypic changes occurring within epithelial cancer cells on culture on a superhydrophobic surface were characterized. hPSCs treated with a Wnt agonist (CHIR) differentiated into mesendodermal precursors completely within 50 hours of treatment, but primarily between 20 and 30 hours of treatment. Significant differences in the levels of phosphorylated β-catenin were observed throughout the differentiation process. PKA phosphorylated β-catenin increased significantly after 20 hours of CHIR treatment and was spatially and temporally correlated with Brachyury expression. Inhibition of PKA along with CHIR led to the attenuation of Brachyury expression. These insightsiii were used to develop a new layout for the Wnt pathway focused on site-specific β-catenin phosphorylation. A mathematical framework for this differentiation was also developed. Vocal fold epithelial barrier characterization has historically been difficult due to the limited viability of ex vivo tissue. Previous hPSC derived tissues were unable to create a confluent cell layer for the accurate characterization of barrier properties. To address these challenges a vocal fold epithelia differentiation protocol was developed for the scalable differentiation on Transwell® inserts and for barrier characterization. The trans-epithelial electrical resistance of the differentiated tissue observed on these inserts was 2556 ± 126 Ω cm 2 and the minimum permeability of Sodium Fluorescein through the differentiated tissue was 8.14 ± 1.95 nm/s. The electrical resistance of 4.5 cm 2 and 1.12 cm 2 inserts were statistically insignificant from each other (p-value=0.83), indicating the scalability of the protocol. It was observed that epithelial cancer cells grown on a water resistant superhydrophobic substrate were up to 80% viable at high densities. Cells grown on this substrate for 12 hours or longer did not adhere to polystyrene unlike cells grown on adherent surfaces or surfaces functionalized to have low cellular attachment. This phenotypic change was also observed with cells grown on a shaken superhydrophobic surface.