Cardiac differentiation in vitro is a complex, stepwise process that is rigidly governed by a subset of transcription factors and signaling cascades. In this study, we investigated the cooperation of cardiac-specific transcription factors Gata4 and Nkx2.5, as well as mitogen-activated protein kinase (MAPK) cascades. P19 embryonic carcinoma cells were induced into spontaneously beating cardiomyocytes utilizing a two-step protocol that comprised an early stage and a late stage of differentiation. During early-stage differentiation in suspension culture, P19 cells aggregated to form embryoid bodies (EBs), and the Gata4 and Nkx2.5 genes were induced. However, Gata4 expressed at the early stage of differentiation was incapable of activating downstream gene expression, as it was localized in the cytoplasm and prone to degradation. After EBs were plated for late-stage differentiation in adherent culture, the MAPK cascades were highly activated and contributed to the activation of Gata4 and Nkx2.5. Specifically, we revealed that p38 signaling participated in regulating the localization and stabilization of Gata4 and Nkx2.5. Additionally, the JNK cascade regulated late-stage cardiac differentiation; JNK kinase reduced Gata4 stabilization and conversely alleviated Nkx2.5 degradation by direct interaction and phosphorylation of Nkx2.5. Finally, we found that the C-terminal domain of Nkx2.5 was required for its stabilization under conditions of oxidative stress and JNK activation. Overall, our results indicated that the induction and activation of Gata4 and Nkx2.5 during early- and late-stage cardiac differentiation was closely associated with the function of the MAPK signaling cascades.