Lipid droplets (LDs) are ubiquitous, neutral lipid storage organelles that act as hubs of metabolic processes. LDs are structurally unique with a hydrophobic core that mainly consists of neutral lipids, sterol esters, and triglycerides, enclosed within a phospholipid monolayer. Nascent LD formation begins with the accumulation of neutral lipids in the endoplasmic reticulum (ER) bilayer. The ER membrane proteins such as seipin, LDAF1, FIT, and MCTPs are reported to play an important role in the formation of nascent LDs. As the LDs grow, they unmix from the highly charged ER membrane to form mature LDs. LD biogenesis is an exciting, emerging research area, and herein, we discuss the recent progress in our understanding of the formation of eukaryotic nascent LDs. We focus on the role of ER membrane shaping proteins such as reticulons and reticulon-like proteins, membrane lipids, and cytoskeleton proteins such as septin in the formation of nascent LDs.