Lecce goalkeeper Wladimiro Falcone, Torino defender Alessandro Buongiorno and Tigre striker Mateo Retegui are all in contention to make their senior international debuts for the Azzurri.
Falcone and Buongiorno were both selected to link up with Mancini's first-team squad for a training camp at the end of last year, after the four-time World Cup winners failed to qualify for the tournament in Qatar.
The duo are now in line to receive their first Azzurri cap along with Retegui, who Mancini has been keeping tabs on for some time.
The 23-year-old striker - who was born in Argentina but is eligible to represent Italy through family descent - has excelled in South America with Tigre, scoring 29 goals in 50 appearances, including six strikes in seven games in 2023.
"We've been following Retegui for a while, he's been playing regularly in the Argentine league for two seasons. He has qualities that we currently lack," Mancini told reporters.
Elsewhere in Mancini's squad, Inter Milan full-back Matteo Darmian has received his first Italy call-up since March 2018, while Lazio defender Alessio Romagnoli has returned for the first time since November 2020.
Arsenal midfielder Jorginho, West Ham United striker Gianluca Scamacca and Leeds United forward Wilfried Gnonto are the only three Premier League players selected in the 30-man squad.
Lazio striker Ciro Immobile recently picked up a hamstring injury and will not be involved during this month's international break, while Napoli's Giacomo Raspadori has been left out having struggled for game time with the Serie A leaders this term.
The Azzurri begin their defence of their European Championship crown when they welcome England to the Stadio Diego Armando Maradona on March 23, before travelling to the Ta' Qali National Stadium to face Malta three days later.
ITALY SQUAD IN FULL:
Goalkeepers: Gianluigi Donnarumma (Paris Saint-Germain), Wladimiro Falcone (Lecce), Alex Meret (Napoli), Ivan Provedel (Lazio)
Defenders: Francesco Acerbi (Inter Milan), Leonardo Bonucci (Juventus), Alessandro Buongiorno (Torino), Matteo Darmian (Inter Milan), Giovanni Di Lorenzo (Napoli), Federico Dimarco (Inter Milan), Alessio Romagnoli (Lazio), Giorgio Scalvini (Atalanta BC), Leonardo Spinazzola (Roma), Rafael Toloi (Atalanta BC)
Midfielders: Nicolo Barella (Inter Milan), Bryan Cristante (Roma), Davide Frattesi (Sassuolo), Jorginho (Arsenal), Lorenzo Pellegrini (Roma), Matteo Pessina (Monza), Sandro Tonali (AC Milan), Marco Verratti (Paris Saint-Germain)
Forwards: Domenico Berardi (Sassuolo), Federico Chiesa (Juventus), Wilfried Gnonto (Leeds United), Vincenzo Grifo (Freiburg), Simone Pafundi (Udinese), Matteo Politano (Napoli), Mateo Retegui (Tigre), Gianluca Scamacca (West Ham United)