During embryonic development, the heart forms from two distinct cell lineages: the first heart field, which forms the cardiac crescent and gives rise to the left ventricle, and the second heart field, which contributes to the formation of the right ventricle and the outflow tract. These cardiac progenitors originate from the mesoderm during gastrulation, particularly from Mesp1-expressing cells. Recent studies have shown that Zic2 and Zic3 are essential cofactors that act with Mesp1 to regulate its transcriptional-factor activity and the early specification of cardiac progenitors. Zic2 and Zic3 are members of the zinc-finger protein family that encode transcription factors involved in the early stages of left-right body axis formation. Mutations in ZIC3 have been identified in patients with X-linked heterotaxy and associated cardiac abnormalities. In mice, Zic3 knockout results in congenital heart defects and heterotaxy. To investigate how the loss of Zic3 affects cardiac cell fate, we used mouse embryonic stem cell (mESC)-derived gastruloids, which recapitulate early developmental processes, as established in our previous study (Argiro et al., 2024). Gastruloids were generated from Zic3-KO mESCs, and beating regions were observed by day 7, indicating that cardiac differentiation can occurr despite the absence of Zic3 expression. Flow cytometry analysis combined with immunofluorescence labeling of cardiac troponin T (cTnT) in cells dissociated from gastruloids confirmed their cardiomyocyte identity. Whole-mount RNA-FISH experiments were also performed on control and Zic3-KO gastruloids collected at day 5. Labeling of left-side polarity markers (Lefty2 and Nodal), the posterior polarity marker (Cyp26a1), and the ventral marker (Shh) revealed defects in axis polarization across several mutant gastruloids. Specifically, we observed a reduction in Nodal-positive cells and altered expression of both Shh and Cyp26a1. In addition, we generated chimeric gastruloids combining wild-type and Zic3-KO cells to study the behavior of mutant cells in a mixed cellular environment. Our findings show that Zic3 is essential for proper cardiac progenitor specification and embryonic axis patterning, as its loss disrupts polarity marker expression despite allowing partial cardiomyocyte differentiation in mES-derived gastruloids.