2.1 Commercially Available Reagents.	See Table 1
2.2 Preparation of Extracellular Matrices (ECMs)	Add the corresponding volume of diluted ECMs to coat different tissue culture plates according to Table 2.
2.2.1 Growth Factor-Reduced Matrigel	Thaw a 10 ml vial of growth factor-reduced matrigel on ice for 1 hour (h) or overnight at 4 °C. Add 10 ml of cold DMEM/F12 medium and mix quickly and thoroughly. Quickly prepare 600 μl aliquots, to be stored at −80 °C.
2.2.2 Diluted Growth Factor-Reduced Matrigel	Thaw a 600 μl aliquot of growth factor-reduced matrigel on ice for 1 h or overnight at 4 °C. Add 50 ml of cold DMEM/F12 medium to a 50 ml falcon tube. Dilute the matrigel aliquot into DMEM/ F12 and quickly mix thoroughly by inverting the falcon tube several times. Cell culture plates are subsequently incubated with matrigel solution to coat surfaces. Coated plates may be stored at 37 °C for up to 5 days prior to cell seeding.
2.2.3 Laminin	A 1 ml vial of laminin is thawed on ice and 50 μl aliquots are prepared for storage at −80 °C. For laminin coating, a 50 μl laminin aliquot is thawed on ice and diluted into a 5 ml solution of PBS+, mixed thoroughly and incubated on tissue culture dish surfaces overnight at 37 °C. Coated dishes may be stored at 4 °C for up to 7 days prior to cell seeding.
2.3 Cell Culture Medium	hiPSCs are maintained in Essential 8 (E8) medium (22). Prepare stock solutions and make aliquots as indicated in Table 3. See Note 1.
2.3.1 hiPSC Culture Medium	Prepare a 500 ml bottle of E8 medium by adding supplements according to Table 4. See Note 2.
2.3.2 Differentiation Initiation Medium: RPMI Medium with B27 Supplement Without Insulin (RB−)	Take a 500 ml bottle of RPMI and add one 10 ml vial of B27 without insulin. Add one 5 ml aliquot of P/S. Mix well. If the medium will be used within 1–10 days, then the bottle can be kept at 4 °C and 50 ml aliquots can be generated to be warmed when needed. Otherwise, 50 ml aliquots should be prepared for storage at −20 °C.
2.3.3 Differentiation Medium: RPMI Medium with B27 Containing Insulin (RB+)	Take a 500 ml bottle of RPMI and add one 10 ml vial of B27 with insulin. Add one 5 ml aliquot of P/S. Mix well. If the medium will be used within 1–10 days, then the bottle can be kept at 4 °C and 50 ml aliquots can be generated to be warmed when needed. Otherwise, 50 ml aliquots should be prepared for storage at −20 °C.
2.3.4 Maturation Medium: DMEM/M199 Medium with B27 Containing Insulin (D/199B+)	Take a fresh bottle of DMEM and remove 140 ml of the medium. Add 125 ml of M199. Add one 10 ml vial of B27 with insulin. Add one 5 ml aliquot of P/S. Mix well. If the medium will be used within 1–10 days, then the bottle can be kept at 4 °C and 50 ml aliquots can be generated to be warmed when needed. Otherwise, 50 ml aliquots should be prepared for storage at −20 °C.
2.4 Small Molecules	For a 10 mM (2,000×) solution, re-suspend 10 mg vial in 3.122 ml of DMSO. Mix well, vortex, and make 25–50 μl aliquots, to be stored at −20 °C. Aliquots may be reused up to three times.
2.4.1 Rock Inhibitor/ Y27632: Rocki
2.4.2 CHIR99021: CH	For a 12 mM (2,000×) solution, re-suspend 25 mg vial in 4.152 ml of DMSO. Mix well, vortex, and make 25–50 μl aliquots, to be stored at −20 °C. Aliquots may be reused up to three times.
2.4.3 IWP-2	For a 5 mM (1,000×) solution, re-suspend 10 mg vial in 4.286 ml of pre-warmed DMSO. Mix well, vortex, and make 25–50 μl aliquots, to be stored at −20 °C. Aliquots may be reused up to three times.
2.5 Dissociation Reagents	Add 500 μl of 0.5 M EDTA to a bottle of PBS−.
2.5.1 hiPSC Dissociation Solution: 0.5 mM EDTA
2.5.2 Collagenase Type II Solution	Cardiomyocytes are dissociated in a solution containing 200 units/ ml of HBSS−. Estimate the required volume of collagenase II solution, and calculate and weigh the mass necessary to reach the target concentration in units/ml. Dissolve into HBSS− and filter sterilize. The solution can be kept at 4 °C for up to 1 week.
2.5.3 Cardiomyocyte Dissociation Solution	Prepare cardiomyocyte dissociation solution immediately before use. For 1 ml cardiomyocyte dissociation solution, prepare the stock solutions in tissue culture-grade water as described in Table 5.
2.6 Buffers	In order to prepare 50 ml of immunofluorescence permeabilization buffer, dilute 100 μl of Triton-X in 50 ml of PBS+, for a final concentration of 0.2 % Triton-X.
2.6.1 Immunofluorescence Permeabilization Buffer
2.6.2 Immunofluorescence Dilution Buffer	In order to prepare 50 ml of immunofluorescence buffer, prepare the stock solutions listed in Table 6 and add the appropriate volumes for the final desired concentrations.
2.6.3 Immunofluorescence Blocking Buffer	In order to prepare 10 ml of immunofluorescence blocking buffer, take 9.5 ml of immunofluorescence dilution buffer and add 500 μl of donkey serum for a final concentration of 5 %.
2.6.4 FACS Dilution Buffer	BD Perm/Wash buffer is sold as a 10× concentrate that must be diluted in PBS− before use. For 10 ml of 1× ready-to-use BD Perm/Wash buffer, add 1 ml of 10× concentrate BD Perm/Wash buffer to 9 ml of PBS−.
2.6.5 FACS Blocking Buffer	For 10 ml of FACS blocking buffer, take 9.5 ml of 1× ready-to-use BD Perm/Wash buffer and add 500 μl of donkey serum for a final concentration of 5 %.