The mechanism of starch granule initiation in chloroplasts is not fully understood. Here, we aimed to build on our recent discovery that PROTEIN TARGETING TO STARCH (PTST) family members, PTST2 and PTST3, are key players in starch granule initiation, by identifying and characterizing additional proteins involved in the process in Arabidopsis thaliana chloroplasts. Using immunoprecipitation and mass spectrometry, we demonstrate that PTST2 interacts with two plastidial coiled-coil proteins. Surprisingly, one of the proteins is the thylakoid-associated MAR BINDING FILAMENT-LIKE PROTEIN1 (MFP1), which was proposed to bind plastid nucleoids. The other protein, MYOSIN-RESEMBLING CHLOROPLAST PROTEIN (MRC), contains long coiled coils and no known domains. Whereas wild-type chloroplasts contained multiple starch granules, only one large granule was observed in most chloroplasts of the mfp1 and mrc mutants. The mfp1 mrc double mutant had a higher proportion of chloroplasts containing no visible granule than either single mutant and accumulated ADP-glucose, the substrate for starch synthesis. PTST2 was partially associated with the thylakoid membranes in wild-type plants, and fluorescently tagged PTST2 was located in numerous discrete patches within the chloroplast in which MFP1 was also located. In the mfp1 mutant, PTST2 was not associated with the thylakoids and formed discrete puncta, suggesting that MFP1 is necessary for normal PTST2 localization. Overall, we reveal that proper granule initiation requires the presence of MFP1 and MRC, and the correct location of PTST2.