(A) In EB, after treatment with the indicated growth factors, mesoderm is induced efficiently from days 1-4 (assessed by mRNA, upper half, blue full line) and cardiomyocytes occur spontaneously from day 6 onward (assessed by mRNA, upper half, red dashed line). For the HCS assay, day 4 EBs were dissociated, plated, and treated with molecules and/or growth factors as depicted in the lower half. (B) Day 4 cells have reached maximum mesoderm levels and were further characterized for early cardiac markers by RT-qPCR for MESP1 and for PDGFRα by flow cytometry (average ± standard deviation, n = 3). (C,D) The HCS assay was screened against a collection of 244 kinase inhibitors (InhibitorSelect) (C) and a collection of 305 pathway modulators (StemSelect) (D) in three concentrations (0.3, 1 and 3μM). All compounds with a Z score larger than 2 are shown for the StemSelect library (D). (E) Fluorescence image of cells expressing the MYH6-mCherry reporter under vehicle or IWR treated cultures.