Recent advancements in single-cell analysis have revolutionized our understanding of cellular heterogeneity, particularly in lipid metabolism. Single-cell lipidomics, enabled by ultra-sensitive mass spectrometry techniques such as Orbitrap and Fourier-transform ion cyclotron resonance (FT-ICR), provides unprecedented insights into lipid-mediated cellular functions. Unlike bulk analyses, single-cell approaches capture real-time metabolic changes, highlighting lipid species' roles in cell differentiation, signal transduction, and disease progression. Mass spectrometry imaging (MSI), including MALDI-MSI and SIMS, further delineates lipid distributions within tissues, revealing spatial heterogeneity critical to cellular function. Emerging evidence suggests that lipid alterations significantly impact developmental mechanisms, stem cell niches, and disease pathogenesis, challenging conventional bulk-level assumptions. However, a key challenge remains in deciphering how lipid networks coordinate cellular differentiation and transcriptional regulation. Future research must integrate lipidomic, proteomic, and genomic data to unravel lipid-mediated signaling and epigenetic modifications. Understanding these dynamics will advance regenerative medicine and therapeutic interventions, enabling precise targeting of lipid-driven pathways in disease contexts.